4′-O-substituted isoindoline derivatives and compositions comprising and methods of using the same

ABSTRACT

Provided are 4′-O substituted isoindoline compounds, and pharmaceutically acceptable salts, solvates, clathrates, stereoisomers, and prodrugs thereof. Methods of use, and pharmaceutical compositions of these compounds are disclosed.

This application is a divisional of co-pending U.S. Application No.13/410,216, filed Mar. 1, 2012, which is a continuation of U.S. patentapplication No. 12,077,715, now U.S. Pat. No. 8,153,659, filed Mar. 19,2008, which claims priority to U.S. Provisional Application No.60/919,323, filed Mar. 20, 2007, entitled “4′-O-Substituted IsoindolineDerivatives And Compositions Comprising And Methods Of Using The Same”,to Ruchelman et al., all of which are incorporated herein by referencein their entireties.

1. FIELD

Provided herein are 4′-O-substituted isoindoline derivatives.Pharmaceutical compositions comprising the compounds and methods fortreating, preventing and managing various disorders using the compoundsand compositions are also disclosed.

2. BACKGROUND

2.1 Pathobiology of Cancer and Other Diseases

Cancer is characterized primarily by an increase in the number ofabnormal cells derived from a given normal tissue, invasion of adjacenttissues by these abnormal cells, or lymphatic or blood-borne spread ofmalignant cells to regional lymph nodes and to distant sites(metastasis). Clinical data and molecular biologic studies indicate thatcancer is a multistep process that begins with minor preneoplasticchanges, which may under certain conditions progress to neoplasia. Theneoplastic lesion may evolve clonally and develop an increasing capacityfor invasion, growth, metastasis, and heterogeneity, especially underconditions in which the neoplastic cells escape the host's immunesurveillance. Roitt, I., Brostoff, J and Kale, D., Immunology,17.1-17.12 (3rd ed., Mosby, St. Louis, Mo., 1993).

There is an enormous variety of cancers which are described in detail inthe medical literature. Examples includes cancer of the lung, colon,rectum, prostate, breast, brain, and intestine. The incidence of cancercontinues to climb as the general population ages, as new cancersdevelop, and as susceptible populations (e.g., people infected with AIDSor excessively exposed to sunlight) grow. However, options for thetreatment of cancer are limited. For example, in the case of bloodcancers (e.g., multiple myeloma), few treatment options are available,especially when conventional chemotherapy fails and bone-marrowtransplantation is not an option. A tremendous demand therefore existsfor new methods and compositions that can be used to treat patients withcancer.

Many types of cancers are associated with new blood vessel formation, aprocess known as angiogenesis. Several of the mechanisms involved intumor-induced angiogenesis have been elucidated. The most direct ofthese mechanisms is the secretion by the tumor cells of cytokines withangiogenic properties. Examples of these cytokines include acidic andbasic fibroblastic growth factor (a,b-FGF), angiogenin, vascularendothelial growth factor (VEGF), and TNF-α. Alternatively, tumor cellscan release angiogenic peptides through the production of proteases andthe subsequent breakdown of the extracellular matrix where somecytokines are stored (e.g., b-FGF). Angiogenesis can also be inducedindirectly through the recruitment of inflammatory cells (particularlymacrophages) and their subsequent release of angiogenic cytokines (e.g.,TNF-α, b-FGF).

A variety of other diseases and disorders are also associated with, orcharacterized by, undesired angiogenesis. For example, enhanced orunregulated angiogenesis has been implicated in a number of diseases andmedical conditions including, but not limited to, ocular neovasculardiseases, choroidal neovascular diseases, retina neovascular diseases,rubeosis (neovascularization of the angle), viral diseases, geneticdiseases, inflammatory diseases, allergic diseases, and autoimmunediseases. Examples of such diseases and conditions include, but are notlimited to: diabetic retinopathy; retinopathy of prematurity; cornealgraft rejection; neovascular glaucoma; retrolental fibroplasia;arthritis; and proliferative vitreoretinopathy.

Accordingly, compounds that can control angiogenesis or inhibit theproduction of certain cytokines, including TNF-α, may be useful in thetreatment and prevention of various diseases and conditions.

2.2 Methods of Treating Cancer

Current cancer therapy may involve surgery, chemotherapy, hormonaltherapy and/or radiation treatment to eradicate neoplastic cells in apatient (see, e.g., Stockdale, 1998, Medicine, vol. 3, Rubenstein andFederman, eds., Chapter 12, Section IV). Recently, cancer therapy couldalso involve biological therapy or immunotherapy. All of theseapproaches pose significant drawbacks for the patient. Surgery, forexample, may be contraindicated due to the health of a patient or may beunacceptable to the patient.

Additionally, surgery may not completely remove neoplastic tissue.Radiation therapy is only effective when the neoplastic tissue exhibitsa higher sensitivity to radiation than normal tissue. Radiation therapycan also often elicit serious side effects. Hormonal therapy is rarelygiven as a single agent. Although hormonal therapy can be effective, itis often used to prevent or delay recurrence of cancer after othertreatments have removed the majority of cancer cells. Biologicaltherapies and immunotherapies are limited in number and may produce sideeffects such as rashes or swellings, flu-like symptoms, including fever,chills and fatigue, digestive tract problems or allergic reactions.

With respect to chemotherapy, there are a variety of chemotherapeuticagents available for treatment of cancer. A majority of cancerchemotherapeutics act by inhibiting DNA synthesis, either directly, orindirectly by inhibiting the biosynthesis of deoxyribonucleotidetriphosphate precursors, to prevent DNA replication and concomitant celldivision. Gilman et al., Goodman and Gilman's: The Pharmacological Basisof Therapeutics, Tenth Ed. (McGraw Hill, N.Y.).

Despite availability of a variety of chemotherapeutic agents,chemotherapy has many drawbacks. Stockdale, Medicine, vol. 3, Rubensteinand Federman, eds., ch. 12, sect. 10, 1998. Almost all chemotherapeuticagents are toxic, and chemotherapy causes significant, and oftendangerous side effects including severe nausea, bone marrow depression,and immunosuppression. Additionally, even with administration ofcombinations of chemotherapeutic agents, many tumor cells are resistantor develop resistance to the chemotherapeutic agents. In fact, thosecells resistant to the particular chemotherapeutic agents used in thetreatment protocol often prove to be resistant to other drugs, even ifthose agents act by different mechanism from those of the drugs used inthe specific treatment. This phenomenon is referred to as pleiotropicdrug or multidrug resistance. Because of the drug resistance, manycancers prove or become refractory to standard chemotherapeutictreatment protocols.

Other diseases or conditions associated with, or characterized by,undesired angiogenesis are also difficult to treat. However, somecompounds such as protamine, hepain and steroids have been proposed tobe useful in the treatment of certain specific diseases. Taylor et al.,Nature 297:307 (1982); Folkman et al., Science 221:719 (1983); and U.S.Pat. Nos. 5,001,116 and 4,994,443.

Still, there is a significant need for safe and effective methods oftreating, preventing and managing cancer and other diseases andconditions, including for diseases that are refractory to standardtreatments, such as surgery, radiation therapy, chemotherapy andhormonal therapy, while reducing or avoiding the toxicities and/or sideeffects associated with the conventional therapies.

3. SUMMARY

Provided herein are 4′-O-substituted isoindoline compounds, andpharmaceutically acceptable salts, solvates (e.g., hydrates), prodrugs,clathrates, or stereoisomers thereof.

Also provided are methods of treating and managing various diseases ordisorders. The methods comprise administering to a patient in need ofsuch treatment or management a therapeutically effective amount of acompound provided herein, or a pharmaceutically acceptable salt,solvate, prodrug, clathrate, or stereoisomer thereof.

Further provided are methods of preventing various diseases anddisorders, which comprise administering to a patient in need of suchprevention a prophylactically effective amount of a compound providedherein, or a pharmaceutically acceptable salt, solvate, prodrug,clathrate, or stereoisomer thereof.

Also provided herein are pharmaceutical compositions, single unit dosageforms, dosing regimens and kits which comprise a compound providedherein, or a pharmaceutically acceptable salt, solvate, prodrug,clathrate, or stereoisomer thereof.

4. DETAILED DESCRIPTION

In one embodiment, provided are isoindoline compounds, andpharmaceutically acceptable salts, solvates, prodrugs, clathrate, andstereoisomers thereof.

In another embodiment, provided are methods of treating, managing, andpreventing various diseases and disorders, which comprises administeringto a patient in need of such treatment or prevention a therapeuticallyor prophylactically effective amount of a compound provided herein, or apharmaceutically acceptable salt, solvate, prodrug, clathrate, orstereoisomer thereof. Examples of diseases and disorders are describedherein.

In other embodiments, a compound provided herein, or a pharmaceuticallyacceptable salt, solvate, prodrug, clathrate, or stereoisomer thereof,is administered in combination with another drug (“second active agent”)or treatment. Second active agents include small molecules and largemolecules (e.g., proteins and antibodies), examples of which areprovided herein, as well as stem cells. Methods, or therapies, that canbe used in combination with the administration of compounds providedherein include, but are not limited to, surgery, blood transfusions,immunotherapy, biological therapy, radiation therapy, and other non-drugbased therapies presently used to treat, prevent or manage variousdisorders described herein.

Also provided are pharmaceutical compositions (e.g., single unit dosageforms) that can be used in the methods provided herein. In oneembodiment, pharmaceutical compositions comprise a compound providedherein, or a pharmaceutically acceptable salt, solvate, prodrug,clathrate, or stereoisomer thereof, and optionally a second activeagent.

4.1 Compounds

In one embodiment, the compounds provided herein for use in thepharmaceutical compositions and methods have formula I:

or a pharmaceutically acceptable salt, solvate, prodrug, clathrate, orstereoisomer thereof, wherein Y is C═O or CH₂, and R¹ is hydrogen,alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl,heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl,arylaminocarbonyl, alkylcarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkoxycarbonyl, cycloalkylcarbonyl,heteroarylcarbonyl or heterocyclylcarbonyl; where R¹ is optionallysubstituted with one or more, in certain embodiments, 1, 2, 3 or 4substituents, one, two or three groups selected from alkoxy, halo,alkyl, carboxy, alkylaminocarbonyl, alkoxycarbonyl, nitro, amine,nitrile, haloalkyl, hydroxy, and alkylsulfonyl.

In one embodiment, Y is C═O. In another embodiment, Y is CH₂.

In certain embodiments, R¹ is alkyl, alkenyl, alkynyl, aryl, aralkyl,cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroarylor heteroarylalkyl, optionally substituted with one or more, in oneembodiment, one, two or three groups selected from alkoxy, halo, alkyland alkylsulfonyl. In one embodiment, R¹ is aryl, aralkyl orheteroarylalkyl. In certain embodiments, the aryl or heteroaryl ring ingroup R¹ is a 5 or 6 membered monocyclic ring. In certain embodiments,the heteroaryl ring in R¹ group is a 5 or 6 membered monocyclic ringcontaining 1-3 heteroatoms selected from O, N and S. In certainembodiments, the aryl or heteroaryl ring in group R¹ is a bicyclic ring.In certain embodiments, the heteroaryl ring contains 1-3 heteroatomsselected from O, N and S and is attached to the alkyl group via a heteroatom in the ring. In certain embodiments, the heteroaryl ring isattached to the alkyl group via a carbon atom in the ring.

In one embodiment, R¹ is phenyl, benzyl, naphthylmethyl, quinolylmethyl,benzofurylmethyl, benzothienylmethyl, furylmethyl or thienylmethyl,optionally substituted with one or more, in one embodiment, one, two orthree groups selected from alkoxy, halo, alkyl and alkylsulfonyl. In oneembodiment, R¹ is optionally substituted with one or two substituentsselected from methoxy, chloro, bromo, fluoro, methyl and methylsulfonyl.

In other embodiments, R¹ is 2-methoxyphenyl, benzyl, 3-chlorobenzyl,4-chlorobenzyl, 3,4-dichlorobenzyl, 3,5-dichlorobenzyl, 3-fluorobenzyl,3-bromobenzyl, 3-methylbenzyl, 4-methylsulfonylbenzyl, 3-methoxybenzyl,naphthylmethyl, 3-quinolylmethyl, 2-quinolylmethyl, 2-benzofurylmethyl,2-benzothienylmethyl, 3-chlorothien-2-ylmethyl,4-fluorobenzothien-2-ylmethyl, 2-furylmethyl, 5-chlorothien-2-ylmethylor 1-naphth-2-ylethyl.

In one embodiment, R¹ is heterocyclyl. In certain embodiments, theheterocyclyl ring in R¹ group is a 5 or 6 membered monocyclic ringcontaining 1-3 heteroatoms selected from O, N and S. In certainembodiments, the heterocyclyl ring in group R¹ is piperidinyl ortetrahydropyranyl.

In certain embodiments, the compounds have formula II:

wherein Y is C═O or CH₂, and R⁵ is aryl or heteroaryl, optionallysubstituted with one, two or three groups selected from alkyl, halo,alkoxy, carboxy, alkylaminocarbonyl, alkoxycarbonyl, nitro, amine,nitrile, haloalkyl, hydroxy, and alkylsulfonyl; n₁ is 0-5, and the othervariables are as described elsewhere herein.

In one embodiment, Y is C═O. In another embodiment, Y is CH₂.

In one embodiment, n₁ is 0 or 1. In certain embodiments, R⁵ is selectedfrom phenyl, naphthyl, furyl, thienyl, benzofuryl, benzothienyl andquinolyl, optionally substituted with one or two groups selected frommethyl, methoxy, chloro, fluoro, bromo and methylsulfonyl. In otherembodiments, R⁵ is phenyl, 3-chlorophenyl, 4-chlorophenyl,3,4-dichlorophenyl, 3,5-dichlorophenyl, 3-fluorophenyl, 3-bromophenyl,3-methylphenyl, 4-methylsulfonylphenyl, 3-methoxyphenyl, naphthyl,3-quinolyl, 2-quinolyl, 2-benzofuryl, 2-benzothienyl,3-chlorothien-2-yl, 4-fluorobenzothien-2-yl, 2-furyl, 5-chlorothien-2-ylor 1-naphth-2-yl.

In one embodiment, n₁ is 0 or 1. In certain embodiments, R⁵ is selectedfrom phenyl, benzyl, naphthyl, furyl, thienyl, benzofuryl, benzothienyland quinolyl, optionally substituted with one or two groups selectedfrom methyl, methoxy, chloro, fluoro, bromo and methylsulfonyl.

In one embodiment, the compounds have formula III

wherein the variables are as described elsewhere herein.

In one embodiment, Y is C═O. In another embodiment, Y is CH₂.

In one embodiment, R⁵ is

Examples include, but are not limited to, those listed below, or apharmaceutically acceptable salt, solvate (e.g., hydrate), prodrug,clathrate, or stereoisomer thereof:

In certain embodiments, the compound is:

In one embodiment, the compound is selected from

As used herein, and unless otherwise specified, the term“pharmaceutically acceptable salt” refers to salts prepared frompharmaceutically acceptable non-toxic acids, including inorganic acidsand organic acids. Suitable non-toxic acids include inorganic andorganic acids such as, but not limited to, acetic, alginic, anthranilic,benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic,formic, fumaric, furoic, gluconic, glutamic, glucuronic, galacturonic,glycidic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic,mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic,phenylacetic, propionic, phosphoric, salicylic, stearic, succinic,sulfanilic, sulfuric, tartaric acid, p-toluenesulfonic and the like. Inone embodiment, suitable are hydrochloric, hydrobromic, phosphoric, andsulfuric acids.

As used herein, and unless otherwise specified, the term “solvate” meansa compound that further includes a stoichiometric or non-stoichiometricamount of solvent bound by non-covalent intermolecular forces. Where thesolvent is water, the solvate is a hydrate.

As used herein, and unless otherwise specified, the term “prodrug” meansa derivative of a compound that can hydrolyze, oxidize, or otherwisereact under biological conditions (in vitro or in vivo) to provide thecompound. Examples of prodrugs include, but are not limited to,compounds that comprise biohydrolyzable moieties such as biohydrolyzableamides, biohydrolyzable esters, biohydrolyzable carbamates,biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzablephosphate analogues. Other examples of prodrugs include compounds thatcomprise —NO, —NO₂, —ONO, or —ONO₂ moieties. Prodrugs can typically beprepared using well-known methods, such as those described in Burger'sMedicinal Chemistry, and Drug Discovery, 172-178, 949-982 (Manfred E.Wolff ed., 5th ed. 1995), and Design of Prodrugs (H. Bundgaard ed.,Elselvier, N.Y. 1985).

As used herein, and unless otherwise specified, the terms“biohydrolyzable carbamate,” “biohydrolyzable carbonate,”“biohydrolyzable ureide” and “biohydrolyzable phosphate” mean acarbamate, carbonate, ureide and phosphate, respectively, of a compoundthat either: 1) does not interfere with the biological activity of thecompound but can confer upon that compound advantageous properties invivo, such as uptake, duration of action, or onset of action; or 2) isbiologically inactive but is converted in vivo to the biologicallyactive compound. Examples of biohydrolyzable carbamates include, but arenot limited to, carbamates that include lower alkylamine, substitutedethylenediamine, aminoacid, hydroxyalkylamine, heterocyclic andheteroaromatic amine, and polyether amine moieties.

As used herein, and unless otherwise specified, the term “stereoisomer”encompasses all enantiomerically/stereomerically pure andenantiomerically/stereomerically enriched compounds provided herein.

As used herein and unless otherwise indicated, the term “stereomericallypure” means a composition that comprises one stereoisomer of a compoundand is substantially free of other stereoisomers of that compound. Forexample, a stereomerically pure composition of a compound having onechiral center will be substantially free of the opposite enantiomer ofthe compound. A stereomerically pure composition of a compound havingtwo chiral centers will be substantially free of other diastereomers ofthe compound. A typical stereomerically pure compound comprises greaterthan about 80% by weight of one stereoisomer of the compound and lessthan about 20% by weight of other stereoisomers of the compound, greaterthan about 90% by weight of one stereoisomer of the compound and lessthan about 10% by weight of the other stereoisomers of the compound,greater than about 95% by weight of one stereoisomer of the compound andless than about 5% by weight of the other stereoisomers of the compound,greater than about 97% by weight of one stereoisomer of the compound andless than about 3% by weight of the other stereoisomers of the compound,greater than about 98% by weight of one stereoisomer of the compound andless than about 2% by weight of the other stereoisomers of the compoundor greater than about 99% by weight of one stereoisomer of the compoundand less than about 1% by weight of the other stereoisomers of thecompound.

As used herein and unless otherwise indicated, the term “stereomericallyenriched” means a composition that comprises greater than about 55% byweight of one stereoisomer of a compound, greater than about 60% byweight of one stereoisomer of a compound, greater than about 70% byweight, or greater than about 80% by weight of one stereoisomer of acompound.

As used herein, and unless otherwise indicated, the term“enantiomerically pure” means a stereomerically pure composition of acompound having one chiral center. Similarly, the term “enantiomericallyenriched” means a stereomerically enriched composition of a compoundhaving one chiral center.

As used herein, and unless otherwise indicated, the term “alkyl” refersto a saturated straight chain or branched hydrocarbon having a number ofcarbon atoms as specified herein. In some embodiments, alkyl groups have1 to 15, 1 to 10, 1 to 6, or 1 to 3 carbon atoms. Representativesaturated straight chain alkyls include -methyl, -ethyl, -n-propyl,-n-butyl, -n-pentyl, and -n-hexyl; while saturated branched alkylsinclude -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl,2-methylbutyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl,4-methylpentyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl,5-methylhexyl, 2,3-dimethylbutyl, and the like. The term “alkyl” alsoencompasses cycloalkyl.

As used herein, alkenyl refers to a straight chain or branchedhydrocarbon containing one or more double bonds. Exemplary alkenylcarbon chains contain from 2 to 20 carbons, and in certain embodiments,contain 1 to 8 double bonds, and the alkenyl carbon chains of 2 to 16carbons, in certain embodiments, contain 1 to 5 double bonds.

As used herein, alkynyl refers to a straight chain or branchedhydrocarbon containing one or more triple bonds. Alkynyl carbon chainsof from 2 to 20 carbons, in certain embodiments, contain 1 to 8 triplebonds, and the alkynyl carbon chains of 2 to 16 carbons, in certainembodiments, contain 1 to 5 triple bonds. Exemplary alkenyl and alkynylgroups herein include, but are not limited to, ethene, propene, butene,pentene, acetylene and hexyne. As used herein, lower alkyl, loweralkenyl, and lower alkynyl refer to carbon chains having from about 1 orabout 2 carbons up to about 6 carbons.

As used herein, and unless otherwise specified, the term “cycloalkyl”means a specie of alkyl, which is cyclic and contains from 3 to 15, 3 to9, 3 to 6, or 3 to 5 carbon atoms, without alternating or resonatingdouble bonds between carbon atoms. It may contain from 1 to 4 rings.Examples of unsubstituted cycloalkyls include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and adamantyl. Acycloalkyl may be substituted with one or more substituents. In someembodiments, a cycloalkyl may be a cycloalkyl fused with aryl orheteroaryl groups.

As used herein and unless otherwise specified, the term“heterocycloalkyl” means a cycloalkyl in which one or more carbon atomsare replaced by heteroatoms such as, but not limited to, N, S, and O. Insome embodiments, a heterocycloalkyl group contains contains from 2 to14, 2 to 8, 2 to 7, 2 to 5, or 2 to 4 carbon atoms. In some embodiments,a heterocycloalkyl may be a heterocycloalkyl fused with aryl orheteroaryl groups.

As used herein, the term “aryl” means a carbocyclic aromatic ringcontaining from 5 to 14 ring atoms. The ring atoms of a carbocyclic arylgroup are all carbon atoms. Aryl ring structures include compoundshaving one or more ring structures such as mono-, bi-, or tricycliccompounds as well as benzo-fused carbocyclic moieties such as5,6,7,8-tetrahydronaphthyl and the like. Specifically, the aryl groupmay be a mono-, bi-, or tricyclic ring. Representative aryl groupsinclude phenyl, anthracenyl, fluorenyl, indenyl, azulenyl, phenanthrenyland naphthyl.

As used herein, “heteroaryl” refers to a monocyclic or multicyclicaromatic ring system, in certain embodiments, of about 5 to about 15members where one or more, in one embodiment 1 to 3, of the atoms in thering system is a heteroatom, that is, an element other than carbon,including but not limited to, nitrogen, oxygen or sulfur. The heteroarylgroup may be optionally fused to a benzene ring. Heteroaryl groupsinclude, but are not limited to, furyl, imidazolyl, indolinyl,pyrrolidinyl, pyrimidinyl, tetrazolyl, thienyl, pyridyl, pyrrolyl,N-methylpyrrolyl, quinolinyl and isoquinolinyl.

As used herein, “heterocyclyl” refers to a monocyclic or multicyclicnon-aromatic ring system, in one embodiment of 3 to 10 members, inanother embodiment of 4 to 7 members, in a further embodiment of 5 to 6members, where one or more, in certain embodiments, 1 to 3, of the atomsin the ring system is a heteroatom, that is, an element other thancarbon, including but not limited to, nitrogen, oxygen or sulfur. Inembodiments where the heteroatom(s) is(are) nitrogen, the nitrogen isoptionally substituted with alkyl, alkenyl, alkynyl, aryl, heteroaryl,aralkyl, heteroaralkyl, cycloalkyl, heterocyclyl, cycloalkylalkyl,heterocyclylalkyl, acyl, guanidino, or the nitrogen may be quaternizedto form an ammonium group where the substituents are selected as above.

As used herein, “aralkyl” refers to an alkyl group in which one of thehydrogen atoms of the alkyl is replaced by an aryl group.

As used herein, “heteroaralkyl” refers to an alkyl group in which one ofthe hydrogen atoms of the alkyl is replaced by a heteroaryl group.

As used herein, “alkylaminocarbonyl” refers to C(O)NHR in which R isalkyl, including lower alkyl. As used herein, “dialkylaminocarbonyl”refers to C(O)NR′R in which R′ and R are independently alkyl, includinglower alkyl; “carboxamide” refers to groups of formula —NR′COR in whichR′ and R are independently alkyl, including lower alkyl.

As used herein, “arylaminocarbonyl” refers to —C(O)NHR in which R isaryl, including lower aryl, such as phenyl.

As used herein, “halo”, “halogen” or “halide” refers to F, Cl, Br or I.

Where the number of any given substituent is not specified (e.g.,“haloalkyl”), there may be one or more substituents present. Forexample, “haloalkyl” may include one or more of the same or differenthalogens.

It should be noted that if there is a discrepancy between a depictedstructure and a name given to that structure, the depicted structure isto be accorded more weight. In addition, if the stereochemistry of astructure or a portion of a structure is not indicated with, forexample, bold or dashed lines, the structure or portion of the structureis to be interpreted as encompassing all stereoisomers of it.

4.2 Methods of Treatment, Prevention and Management

Provided herein are methods of treating, preventing, and/or managingvarious diseases or disorders using a compound provided herein, or apharmaceutically acceptable salt, solvate (e.g., hydrate), prodrug,clathrate, or stereoisomer thereof.

Examples of diseases or disorders include, but are not limited to,cancer, disorders associated with angiogenesis, pain including, but notlimited to, Complex Regional Pain Syndrome (“CRPS”), MacularDegeneration (“MD”) and related syndromes, skin diseases, pulmonarydisorders, asbestos-related disorders, parasitic diseases,immunodeficiency disorders, CNS disorders, CNS injury, atherosclerosisand related disorders, dysfunctional sleep and related disorders,hemoglobinopathy and related disorders (e.g., anemia), TNFα relateddisorders, and other various diseases and disorders.

As used herein, and unless otherwise specified, the terms “treat,”“treating” and “treatment” refer to the eradication or amelioration of adisease or disorder, or of one or more symptoms associated with thedisease or disorder. In certain embodiments, the terms refer tominimizing the spread or worsening of the disease or disorder resultingfrom the administration of one or more prophylactic or therapeuticagents to a subject with such a disease or disorder.

As used herein, unless otherwise specified, the term “preventing” refersto the treatment with or administration of a compound provided herein,with or without other additional active compound, prior to the onset ofsymptoms, particularly to patients at risk of cancer and/or otherdisorders described herein. The term “prevention” includes theinhibition or reduction of a symptom of the particular disease. Patientswith familial history of a disease in particular are candidates forpreventive regimens in certain embodiments. In addition, patients whohave a history of recurring symptoms are also potential candidates forthe prevention. In this regard, the term “prevention” may beinterchangeably used with the term “prophylactic treatment.”

As used herein, and unless otherwise specified, the terms “manage,”“managing” and “management” refer to preventing or slowing theprogression, spread or worsening of a disease or disorder, or of one ormore symptoms thereof. In certain cases, the beneficial effects that asubject derives from a prophylactic or therapeutic agent do not resultin a cure of the disease or disorder.

As used herein, and unless otherwise specified, a “therapeuticallyeffective amount” of a compound is an amount sufficient to provide atherapeutic benefit in the treatment or management of a disease ordisorder, or to delay or minimize one or more symptoms associated withthe disease or disorder. A therapeutically effective amount of acompound means an amount of therapeutic agent, alone or in combinationwith other therapies, which provides a therapeutic benefit in thetreatment or management of the disease or disorder. The term“therapeutically effective amount” can encompass an amount that improvesoverall therapy, reduces or avoids symptoms or causes of disease ordisorder, or enhances the therapeutic efficacy of another therapeuticagent.

As used herein, and unless otherwise specified, a “prophylacticallyeffective amount” of a compound is an amount sufficient to inhibit orreduce a symptom of a disease or to prevent recurrence of a disease. Aprophylactically effective amount of a compound means an amount oftherapeutic agent, alone or in combination with other agents, whichprovides a prophylactic benefit in the inhibition or reduction of asymptom of a disease or recurrence of a disease. The term“prophylactically effective amount” can encompass an amount thatimproves overall prophylaxis or enhances the prophylactic efficacy ofanother prophylactic agent.

Examples of cancer and precancerous conditions include, but are notlimited to, those described in U.S. Pat. Nos. 6,281,230 and 5,635,517 toMuller et al., in various U.S. patent publications to Zeldis, includingpublication nos. 2004/0220144A1, published Nov. 4, 2004 (Treatment ofMyelodysplastic Syndrome); 2004/0029832A1, published Feb. 12, 2004(Treatment of Various Types of Cancer); and 2004/0087546, published May6, 2004 (Treatment of Myeloproliferative Diseases). Examples alsoinclude those described in WO 2004/103274, published Dec. 2, 2004. Allof these references are incorporated herein in their entireties byreference.

Specific examples of cancer include, but are not limited to, cancers ofthe skin, such as melanoma; lymph node; breast; cervix; uterus;gastrointestinal tract; lung; ovary; prostate; colon; rectum; mouth;brain; head and neck; throat; testes; kidney; pancreas; bone; spleen;liver; bladder; larynx; nasal passages; and AIDS-related cancers. Thecompounds are also useful for treating cancers of the blood and bonemarrow, such as multiple myeloma and acute and chronic leukemias, forexample, lymphoblastic, myelogenous, lymphocytic, and myelocyticleukemias. The compounds provided herein can be used for treating,preventing or managing either primary or metastatic tumors.

Other specific cancers include, but are not limited to, advancedmalignancy, amyloidosis, neuroblastoma, meningioma, hemangiopericytoma,multiple brain metastase, glioblastoma multiforms, glioblastoma, brainstem glioma, poor prognosis malignant brain tumor, malignant glioma,recurrent malignant glioma, anaplastic astrocytoma, anaplasticoligodendroglioma, neuroendocrine tumor, rectal adenocarcinoma, Dukes C& D colorectal cancer, unresectable colorectal carcinoma, metastatichepatocellular carcinoma, Kaposi's sarcoma, karotype acute myeloblasticleukemia, chronic lymphocytic leukemia (CLL), Hodgkin's lymphoma,non-Hodgkin's lymphoma, cutaneous T-Cell lymphoma, cutaneous B-Celllymphoma, diffuse large B-Cell lymphoma, low grade follicular lymphoma,metastatic melanoma (localized melanoma, including, but not limited to,ocular melanoma), malignant mesothelioma, malignant pleural effusionmesothelioma syndrome, peritoneal carcinoma, papillary serous carcinoma,gynecologic sarcoma, soft tissue sarcoma, scleroderma, cutaneousvasculitis, Langerhans cell histiocytosis, leiomyosarcoma,fibrodysplasia ossificans progressive, hormone refractory prostatecancer, resected high-risk soft tissue sarcoma, unresectablehepatocellular carcinoma, Waldenstrom's macroglobulinemia, smolderingmyeloma, indolent myeloma, fallopian tube cancer, androgen independentprostate cancer, androgen dependent stage 1V non-metastatic prostatecancer, hormone-insensitive prostate cancer, chemotherapy-insensitiveprostate cancer, papillary thyroid carcinoma, follicular thyroidcarcinoma, medullary thyroid carcinoma, and leiomyoma. In a specificembodiment, the cancer is metastatic. In another embodiment, the canceris refractory or resistance to chemotherapy or radiation.

In one embodiment, provided herein are methods of treating, preventingor managing various forms of leukemias such as chronic lymphocyticleukemia, chronic myelocytic leukemia, acute lymphoblastic leukemia,acute myelogenous leukemia and acute myeloblastic leukemia, includingleukemias that are relapsed, refractory or resistant, as disclosed inU.S. publication no. 2006/0030594, published Feb. 9, 2006, which isincorporated in its entirety by reference.

The term “leukemia” refers malignant neoplasms of the blood-formingtissues. The leukemia includes, but is not limited to, chroniclymphocytic leukemia, chronic myelocytic leukemia, acute lymphoblasticleukemia, acute myelogenous leukemia and acute myeloblastic leukemia.The leukemia can be relapsed, refractory or resistant to conventionaltherapy. The term “relapsed” refers to a situation where patients whohave had a remission of leukemia after therapy have a return of leukemiacells in the marrow and a decrease in normal blood cells. The term“refractory or resistant” refers to a circumstance where patients, evenafter intensive treatment, have residual leukemia cells in their marrow.

In another embodiment, provided herein are methods of treating,preventing or managing various types of lymphomas, includingNon-Hodgkin's lymphoma (NHL). The term “lymphoma” refers a heterogenousgroup of neoplasms arising in the reticuloendothelial and lymphaticsystems. “NHL” refers to malignant monoclonal proliferation of lymphoidcells in sites of the immune system, including lymph nodes, bone marrow,spleen, liver and gastrointestinal tract. Examples of NHL include, butare not limited to, mantle cell lymphoma (MCL), lymphocytic lymphoma ofintermediate differentiation, intermediate lymphocytic lymphoma (ILL),diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocyticlymphoma, diffuse small-cleaved cell lymphoma (DSCCL), follicularlymphoma, and any type of the mantle cell lymphomas that can be seenunder the microscope (nodular, diffuse, blastic and mentle zonelymphoma).

Examples of diseases and disorders associated with, or characterized by,undesired angiogenesis include, but are not limited to, inflammatorydiseases, autoimmune diseases, viral diseases, genetic diseases,allergic diseases, bacterial diseases, ocular neovascular diseases,choroidal neovascular diseases, retina neovascular diseases, andrubeosis (neovascularization of the angle). Specific examples of thediseases and disorders associated with, or characterized by, undesiredangiogenesis include, but are not limited to, arthritis, endometriosis,Crohn's disease, heart failure, advanced heart failure, renalimpairment, endotoxemia, toxic shock syndrome, osteoarthritis,retrovirus replication, wasting, meningitis, silica-induced fibrosis,asbestos-induced fibrosis, veterinary disorder, malignancy-associatedhypercalcemia, stroke, circulatory shock, periodontitis, gingivitis,macrocytic anemia, refractory anemia, and 5q-deletion syndrome.

Examples of pain include, but are not limited to those described in U.S.patent publication no. 2005/0203142, published Sep. 15, 2005, which isincorporated herein by reference. Specific types of pain include, butare not limited to, nociceptive pain, neuropathic pain, mixed pain ofnociceptive and neuropathic pain, visceral pain, migraine, headache andpost-operative pain.

Examples of nociceptive pain include, but are not limited to, painassociated with chemical or thermal burns, cuts of the skin, contusionsof the skin, osteoarthritis, rheumatoid arthritis, tendonitis, andmyofascial pain.

Examples of neuropathic pain include, but are not limited to, CRPS typeI, CRPS type II, reflex sympathetic dystrophy (RSD), reflexneurovascular dystrophy, reflex dystrophy, sympathetically maintainedpain syndrome, causalgia, Sudeck atrophy of bone, algoneurodystrophy,shoulder hand syndrome, post-traumatic dystrophy, trigeminal neuralgia,post herpetic neuralgia, cancer related pain, phantom limb pain,fibromyalgia, chronic fatigue syndrome, spinal cord injury pain, centralpost-stroke pain, radiculopathy, diabetic neuropathy, post-stroke pain,luetic neuropathy, and other painful neuropathic conditions such asthose induced by drugs such as vincristine and velcade.

As used herein, the terms “complex regional pain syndrome,” “CRPS” and“CRPS and related syndromes” mean a chronic pain disorder characterizedby one or more of the following: pain, whether spontaneous or evoked,including allodynia (painful response to a stimulus that is not usuallypainful) and hyperalgesia (exaggerated response to a stimulus that isusually only mildly painful); pain that is disproportionate to theinciting event (e.g., years of severe pain after an ankle sprain);regional pain that is not limited to a single peripheral nervedistribution; and autonomic dysregulation (e.g., edema, alteration inblood flow and hyperhidrosis) associated with trophic skin changes (hairand nail growth abnormalities and cutaneous ulceration).

Examples of MD and related syndromes include, but are not limited to,those described in U.S. patent publication no. 2004/0091455, publishedMay 13, 2004, which is incorporated herein by reference. Specificexamples include, but are not limited to, atrophic (dry) MD, exudative(wet) MD, age-related maculopathy (ARM), choroidal neovascularisation(CNVM), retinal pigment epithelium detachment (PED), and atrophy ofretinal pigment epithelium (RPE).

Examples of skin diseases include, but are not limited to, thosedescribed in U.S. publication no. 2005/0214328A1, published Sep. 29,2005, which is incorporated herein by reference. Specific examplesinclude, but are not limited to, keratoses and related symptoms, skindiseases or disorders characterized with overgrowths of the epidermis,acne, and wrinkles.

As used herein, the term “keratosis” refers to any lesion on theepidermis marked by the presence of circumscribed overgrowths of thehorny layer, including but not limited to actinic keratosis, seborrheickeratosis, keratoacanthoma, keratosis follicularis (Darier disease),inverted follicular keratosis, palmoplantar keratoderma (PPK, keratosispalmaris et plantaris), keratosis pilaris, and stucco keratosis. Theterm “actinic keratosis” also refers to senile keratosis, keratosissenilis, verruca senilis, plana senilis, solar keratosis, keratoderma orkeratoma. The term “seborrheic keratosis” also refers to seborrheicwart, senile wart, or basal cell papilloma. Keratosis is characterizedby one or more of the following symptoms: rough appearing, scaly,erythematous papules, plaques, spicules or nodules on exposed surfaces(e.g., face, hands, ears, neck, legs and thorax), excrescences ofkeratin referred to as cutaneous horns, hyperkeratosis, telangiectasias,elastosis, pigmented lentigines, acanthosis, parakeratosis,dyskeratoses, papillomatosis, hyperpigmentation of the basal cells,cellular atypia, mitotic figures, abnormal cell-cell adhesion, denseinflammatory infiltrates and small prevalence of squamous cellcarcinomas.

Examples of skin diseases or disorders characterized with overgrowths ofthe epidermis include, but are not limited to, any conditions, diseasesor disorders marked by the presence of overgrowths of the epidermis,including but not limited to, infections associated with papillomavirus, arsenical keratoses, sign of Leser-Trélat, warty dyskeratoma(WD), trichostasis spinulosa (TS), erythrokeratodermia variabilis (EKV),ichthyosis fetalis (harlequin ichthyosis), knuckle pads, cutaneousmelanoacanthoma, porokeratosis, psoriasis, squamous cell carcinoma,confluent and reticulated papiliomatosis (CRP), acrochordons, cutaneoushorn, cowden disease (multiple hamartoma syndrome), dermatosis papulosanigra (DPN), epidermal nevus syndrome (ENS), ichthyosis vulgaris,molluscum contagiosum, prurigo nodularis, and acanthosis nigricans (AN).

Examples of pulmonary disorders include, but are not limited to, thosedescribed in U.S. publication no. 2005/0239842A1, published Oct. 27,2005, which is incorporated herein by reference. Specific examplesinclude pulmonary hypertension and related disorders. Examples ofpulmonary hypertension and related disorders include, but are notlimited to: primary pulmonary hypertension (PPH); secondary pulmonaryhypertension (SPH); familial PPH; sporadic PPH; precapillary pulmonaryhypertension; pulmonary arterial hypertension (PAH); pulmonary arteryhypertension; idiopathic pulmonary hypertension; thrombotic pulmonaryarteriopathy (TPA); plexogenic pulmonary arteriopathy; functionalclasses I to IV pulmonary hypertension; and pulmonary hypertensionassociated with, related to, or secondary to, left ventriculardysfunction, mitral valvular disease, constrictive pericarditis, aorticstenosis, cardiomyopathy, mediastinal fibrosis, anomalous pulmonaryvenous drainage, pulmonary venoocclusive disease, collagen vasulardisease, congenital heart disease, HIV virus infection, drugs and toxinssuch as fenfluramines, congenital heart disease, pulmonary venoushypertension, chronic obstructive pulmonary disease, interstitial lungdisease, sleep-disordered breathing, alveolar hypoventilation disorder,chronic exposure to high altitude, neonatal lung disease,alveolar-capillary dysplasia, sickle cell disease, other coagulationdisorder, chronic thromboemboli, connective tissue disease, lupusincluding systemic and cutaneous lupus, schistosomiasis, sarcoidosis orpulmonary capillary hemangiomatosis.

Examples of asbestos-related disorders include, but not limited to,those described in U.S. publication no. 2005/0100529, published May 12,2005, which is incorporated herein by reference. Specific examplesinclude, but are not limited to, mesothelioma, asbestosis, malignantpleural effusion, benign exudative effusion, pleural plaques, pleuralcalcification, diffuse pleural thickening, rounded atelectasis, fibroticmasses, and lung cancer.

Examples of parasitic diseases include, but are not limited to, thosedescribed in U.S. publication no. 2006/0154880, published Jul. 13, 2006,which is incorporated herein by reference. Parasitic diseases includediseases and disorders caused by human intracellular parasites such as,but not limited to, P. falcifarium, P. ovale, P. vivax, P. malariae, L.donovari, L. infantum, L. aethiopica, L. major, L. tropica, L. mexicana,L. braziliensis, T. Gondii, B. microti, B. divergens, B. coli, C.parvum, C. cayetanensis, E. histolytica, L. belli, S. mansonii, S.haematobium, Trypanosoma ssp., Toxoplasma ssp., and O. volvulus. Otherdiseases and disorders caused by non-human intracellular parasites suchas, but not limited to, Babesia bovis, Babesia canis, Banesia Gibsoni,Besnoitia darlingi, Cytauxzoon felis, Eimeria ssp., Hammondia ssp., andTheileria ssp., are also encompassed. Specific examples include, but arenot limited to, malaria, babesiosis, trypanosomiasis, leishmaniasis,toxoplasmosis, meningoencephalitis, keratitis, amebiasis, giardiasis,cryptosporidiosis, isosporiasis, cyclosporiasis, microsporidiosis,ascariasis, trichuriasis, ancylostomiasis, strongyloidiasis,toxocariasis, trichinosis, lymphatic filariasis, onchocerciasis,filariasis, schistosomiasis, and dermatitis caused by animalschistosomes.

Examples of immunodeficiency disorders include, but are not limited to,those described in U.S. application Ser. No. 11/289,723, filed Nov. 30,2005. Specific examples include, but not limited to, adenosine deaminasedeficiency, antibody deficiency with normal or elevated Igs,ataxia-tenlangiectasia, bare lymphocyte syndrome, common variableimmunodeficiency, Ig deficiency with hyper-IgM, Ig heavy chaindeletions, IgA deficiency, immunodeficiency with thymoma, reticulardysgenesis, Nezelof syndrome, selective IgG subclass deficiency,transient hypogammaglobulinemia of infancy, Wistcott-Aldrich syndrome,X-linked agammaglobulinemia, X-linked severe combined immunodeficiency.

Examples of CNS disorders include, but are not limited to, thosedescribed in U.S. publication no. 2005/0143344, published Jun. 30, 2005,which is incorporated herein by reference. Specific examples include,but are not limited to, include, but are not limited to, AmyotrophicLateral Sclerosis, Alzheimer Disease, Parkinson Disease, Huntington'sDisease, Multiple Sclerosis other neuroimmunological disorders such asTourette Syndrome, delerium, or disturbances in consciousness that occurover a short period of time, and amnestic disorder, or discreet memoryimpairments that occur in the absence of other central nervous systemimpairments.

Examples of CNS injuries and related syndromes include, but are notlimited to, those described in U.S. publication no. 2006/0122228,published Jun. 8, 2006, which is incorporated herein by reference.Specific examples include, but are not limited to, CNS injury/damage andrelated syndromes, include, but are not limited to, primary braininjury, secondary brain injury, traumatic brain injury, focal braininjury, diffuse axonal injury, head injury, concussion, post-concussionsyndrome, cerebral contusion and laceration, subdural hematoma,epidermal hematoma, post-traumatic epilepsy, chronic vegetative state,complete SCI, incomplete SCI, acute SCI, subacute SCI, chronic SCI,central cord syndrome. Brown-Sequard syndrome, anterior cord syndrome,conus medullaris syndrome, cauda equina syndrome, neurogenic shock,spinal shock, altered level of consciousness, headache, nausea, emesis,memory loss, dizziness, diplopia, blurred vision, emotional lability,sleep disturbances, irritability, inability to concentrate, nervousness,behavioral impairment, cognitive deficit, and seizure.

Other disease or disorders include, but not limited to, viral, genetic,allergic, and autoimmune diseases. Specific examples include, but notlimited to, HIV, hepatitis, adult respiratory distress syndrome, boneresorption diseases, chronic pulmonary inflammatory diseases,dermatitis, cystic fibrosis, septic shock, sepsis, endotoxic shock,hemodynamic shock, sepsis syndrome, post ischemic reperfusion injury,meningitis, psoriasis, fibrotic disease, cachexia, graft versus hostdisease, graft rejection, auto-immune disease, rheumatoid spondylitis,Crohn's disease, ulcerative colitis, inflammatory-bowel disease,multiple sclerosis, systemic lupus erythrematosus, ENL in leprosy,radiation damage, cancer, asthma, or hyperoxic alveolar injury.

Examples of atherosclerosis and related conditions include, but are notlimited to, those disclosed in U.S. publication no. 2002/0054899,published May 9, 2002, which is incorporated herein by reference.Specific examples include, but are not limited to, all forms ofconditions involving atherosclerosis, including restenosis aftervascular intervention such as angioplasty, stenting, atherectomy andgrafting. All forms of vascular intervention are contemplated herein,including diseases of the cardiovascular and renal system, such as, butnot limited to, renal angioplasty, percutaneous coronary intervention(PCI), percutaneous transluminal coronary angioplasty (PTCA), carotidpercutaneous transluminal angioplasty (PTA), coronary by-pass grafting,angioplasty with stent implantation, peripheral percutaneoustransluminal intervention of the iliac, femoral or popliteal arteries,and surgical intervention using impregnated artificial grafts. Thefollowing chart provides a listing of the major systemic arteries thatmay be in need of treatment, all of which are contemplated herein:

TABLE 1 Artery Body Areas Supplied Axillary Shoulder and axilla BrachialUpper arm Brachiocephalic Head, neck, and arm Celiac Divides into leftgastric, splenic, and hepatic arteries Common carotid Neck Common iliacDivides into external and internal iliac arteries Coronary Heart Deepfemoral Thigh Digital Fingers Dorsalis pedis Foot External carotid Neckand external head regions External iliac Femoral artery Femoral ThighGastric Stomach Hepatic Liver, gallbladder, pancreas, and duodenuminferior mesenteric Descending colon, rectum, and pelvic wall Internalcarotid Neck and internal head regions Internal iliac Rectum, urinarybladder, external genitalia, buttocks Left gastric muscles, uterus andvagina Middle sacral Esophagus and stomach Ovarian Sacrum Palmar archOvaries Peroneal Hand Popliteal Calf Posterior tibial Knee PulmonaryCalf Radial Lungs Renal Forearm Splenic Kidney Subclavian Stomach,pancreas, and spleen Superior mesenteric Shoulder Testicular Pancreas,small intestine, ascending and transverse colon Ulnar Testes Forearm

Examples of dysfunctional sleep and related syndromes include, but arenot limited to, those disclosed in U.S. publication no. 2005/0222209A1,published Oct. 6, 2005, which is incorporated herein by reference.Specific examples include, but are not limited to, snoring, sleep apnea,insomnia, narcolepsy, restless leg syndrome, sleep terrors, sleepwalking sleep eating, and dysfunctional sleep associated with chronicneurological or inflammatory conditions. Chronic neurological orinflammatory conditions, include, but are not limited to, ComplexRegional Pain Syndrome, chronic low back pain, musculoskeletal pain,arthritis, radiculopathy, pain associated with cancer, fibromyalgia,chronic fatigue syndrome, visceral pain, bladder pain, chronicpancreatitis, neuropathies (diabetic, post-herpetic, traumatic orinflammatory), and neurodegenerative disorders such as Parkinson'sDisease, Alzheimer's Disease, amyotrophic lateral sclerosis, multiplesclerosis, Huntington's Disease, bradykinesia; muscle rigidity;parkinsonian tremor; parkinsonian gait; motion freezing; depression;defective long-term memory, Rubinstein-Taybi syndrome (RTS); dementia;postural instability; hypokinetic disorders; synuclein disorders;multiple system atrophies; striatonigral degeneration;olivopontocerebellar atrophy; Shy-Drager syndrome; motor neuron diseasewith parkinsonian features; Lewy body dementia; Tau pathology disorders;progressive supranuclear palsy; corticobasal degeneration;frontotemporal dementia; amyloid pathology disorders; mild cognitiveimpairment; Alzheimer disease with parkinsonism; Wilson disease;Hallervorden-Spatz disease; Chediak-Hagashi disease; SCA-3spinocerebellar ataxia; X-linked dystonia parkinsonism; prion disease;hyperkinetic disorders; chorea; ballismus; dystonia tremors; AmyotrophicLateral Sclerosis (ALS); CNS trauma and myoclonus.

Examples of hemoglobinopathy and related disorders include, but are notlimited to, those described in U.S. publication no. 2005/0143420A1,published Jun. 30, 2005, which is incorporated herein by reference.Specific examples include, but are not limited to, hemoglobinopathy,sickle cell anemia, and any other disorders related to thedifferentiation of CD34+ cells.

Examples of TNFα related disorders include, but are not limited to,those described in WO 98/03502 and WO 98/54170, both of which areincorporated herein in their entireties by reference. Specific examplesinclude, but are not limited to: endotoxemia or toxic shock syndrome;cachexia; adult respiratory distress syndrome; bone resorption diseasessuch as arthritis; hypercalcemia; Graft versus Host Reaction; cerebralmalaria; inflammation; tumor growth; chronic pulmonary inflammatorydiseases; reperfusion injury; myocardial infarction; stroke; circulatoryshock; rheumatoid arthritis; Crohn's disease; HIV infection and AIDS;other disorders such as rheumatoid arthritis, rheumatoid spondylitis,osteoarthritis, psoriatic arthritis and other arthritic conditions,septic shock, septis, endotoxic shock, graft versus host disease,wasting, Crohn's disease, ulcerative colitis, multiple sclerosis,systemic lupus erythromatosis, ENL in leprosy, HIV, AIDS, andopportunistic infections in AIDS; disorders such as septic shock,sepsis, endotoxic shock, hemodynamic shock and sepsis syndrome, postischemic reperfusion injury, malaria, mycobacterial infection,meningitis, psoriasis, congestive heart failure, fibrotic disease,cachexia, graft rejection, oncogenic or cancerous conditions, asthma,autoimmune disease, radiation damages, and hyperoxic alveolar injury;viral infections, such as those caused by the herpes viruses; viralconjunctivitis; or atopic dermatitis.

In other embodiments, the use of compounds provided herein in variousimmunological applications, i.e., use of compounds provided herein incombination with a vaccination, for example, as vaccine adjuvant.Although any methods and manners of use of compounds provided herein incombination with a vaccine are contemplated herein, a non-limitingexample of such uses is the use of compounds provided herein as vaccineadjuvants, according to the administration regimens disclosed in U.S.Provisional Application No. 60/712,823, filed Sep. 1, 2005, which isincorporated herein in its entirety by reference. These embodiments alsorelate to the uses of compounds provided herein in combination withvaccines to treat or prevent cancer or infectious diseases, and othervarious uses of compounds provided herein, such as, but not limited to,reduction or desensitization of allergic reactions.

Doses of a compound provided herein, or a pharmaceutically acceptablesalt, solvate, clathrate, stereoisomer or prodrug thereof, varydepending on factors such as: specific indication to be treated,prevented, or managed; age and condition of a patient; and amount ofsecond active agent used, if any. Generally, a compound provided herein,or a pharmaceutically acceptable salt, solvate, clathrate, stereoisomeror prodrug thereof, may be used in an amount of from about 0.1 mg toabout 500 mg per day, and can be adjusted in a conventional fashion(e.g., the same amount administered each day of the treatment,prevention or management period), in cycles (e.g., one week on, one weekoff), or in an amount that increases or decreases over the course oftreatment, prevention, or management. In other embodiments, the dose canbe from about 1 mg to about 300 mg, from about 0.1 mg to about 150 mg,from about 1 mg to about 200 mg, from about 10 mg to about 100 mg, fromabout 0.1 mg to about 50 mg, from about 1 mg to about 50 mg, from about10 mg to about 50 mg, from about 20 mg to about 30 mg, or from about 1mg to about 20 mg.

4.3 Second Active Agents

A compound provided herein, or a pharmaceutically acceptable salt,solvate, prodrug, clathrate, or stereoisomer thereof, can be combinedwith other pharmacologically active compounds (“second active agents”)in methods and compositions provided herein. Certain combinations maywork synergistically in the treatment of particular types diseases ordisorders, and conditions and symptoms associated with such diseases ordisorders. A compound provided herein, or a pharmaceutically acceptablesalt, solvate, clathrate, stereoisomer or prodrug thereof, can also workto alleviate adverse effects associated with certain second activeagents, and vice versa.

One or more second active ingredients or agents can be used in themethods and compositions provided herein. Second active agents can belarge molecules (e.g., proteins) or small molecules (e.g., syntheticinorganic, organometallic, or organic molecules).

Examples of large molecule active agents include, but are not limitedto, hematopoietic growth factors, cytokines, and monoclonal andpolyclonal antibodies. Specific examples of the active agents areanti-CD40 monoclonal antibodies (such as, for example, SGN-40); histonedeacetylyase inhibitors (such as, for example. SAHA and LAQ 824);heat-shock protein-90 inhibitors (such as, for example, 17-AAG);insulin-like growth factor-1 receptor kinase inhibitors; vascularendothelial growth factor receptor kinase inhibitors (such as, forexample, PTK787); insulin growth factor receptor inhibitors;lysophosphatidic acid acyltransrerase inhibitors; 1 kB kinaseinhibitors; p38MAPK inhibitors; EGFR inhibitors (such as, for example,gefitinib and erlotinib HCL); HER-2 antibodies (such as, for example,trastuzumab (Herceptin®) and pertuzumab (Omnitarg™)); VEGFR antibodies(such as, for example, bevacizumab (Avastin™)); VEGFR inhibitors (suchas, for example, flk-1 specific kinase inhibitors, SU5416 andptk787/zk222584); P13K inhibitors (such as, for example, wortmannin);C-Met inhibitors (such as, for example, PHA-665752); monoclonalantibodies (such as, for example, rituximab (Rituxan®), tositumomab(Bexxar®), edrecolomab (Panorex®) and G250); and anti-TNF-α antibodies.Examples of small molecule active agents include, but are not limitedto, anticancer agents and antibiotics (e.g., clarithromycin).

Specific second active compounds that can be combined with compoundsprovided herein vary depending on the specific indication to be treated,prevented or managed.

For instance, for the treatment, prevention or management of cancer,second active agents include, but are not limited to: semaxanib;cyclosporin; etanercept; doxycyc line; bortezomib; lapatinib (Tykerb®);acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin;aldesleukin; altretamine; ambomycin; ametantrone acetate; amsacrine;anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa;azotomycin; batimastat; benzodepa; bicalutamide; bisantrenehydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate;brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone;caracemide; carbetimer; carboplatin; carmustine; carubicinhydrochloride; carzelesin; cedefingol; celecoxib; chlorambucfl;cirolemycm; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide;cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride;decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate;diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride;droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin;edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin;enpromate; epipropidine; epirubicin hydrochloride; erbulozole;esorubicin hydrochloride; estramustine; estramustine phosphate sodium;etanidazole; etoposide; etoposide phosphate; etoprine; fadrozolehydrochloride; fazarabine; fenretinide; floxuridine; fludarabinephosphate; fluorouracil; fluorocitabine; fosquidone; fostriecin sodium;gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicinhydrochloride; ifosfamide; ilmofosine; iproplatin; irinotecan;irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolideacetate; liarozole hydrochloride; lometrexol sodium; lomustine;losoxantrone hydrochloride; masoprocol; maytansine; mechlorethaminehydrochloride; megestrol acetate; melengestrol acetate; melphalan;menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine;meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin;mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolicacid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel;pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; safingol; safingol hydrochloride; semustine;simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; taxotere; tegafur;teloxantrone hydrochloride; temoporfin; teniposide; teroxirone;testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin;tirapazamine; toremifene citrate; trestolone acetate; triciribinephosphate; trimetrexate; trimetrexate glucuronate; triptorelin;tubulozole hydrochloride; uracil mustard; uredepa; vapreotide;verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; and zorubicinhydrochloride.

Other second agents include, but are not limited to: 20-epi-1,25dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin;acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists;altretamine; ambamustine; amidox; amifostine; aminolevulinic acid;amrubicin; amsacrine; anagrelide; anastrozole; andrographolide;angiogenesis inhibitors; antagonist D; antagonist G; antarelix;anti-dorsalizing morphogenetic protein-1; antiandrogen, prostaticcarcinoma; antiestrogen; antineoplaston; antisense oligonucleotides;aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators;apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine;atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3;azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol;batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine;beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid;bFGF inhibitor; bicalutamide; bisantrene; bisaziridinyispermine;bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane;buthionine sulfoximine; calcipotriol; calphostin C; camptothecinderivatives; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; col lismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel;docosanol; dolasetron; doxifluridine; doxorubicin; droloxifene;dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine;edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride;estramustine analogue; estrogen agonists; estrogen antagonists;etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine;fenretinide; filgrastim; finasteride; flavopiridol; flezelastine;fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex;formestane; fostriecin; fotemustine; gadolinium texaphyrin; galliumnitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;glutathione inhibitors; hepsulfam; heregulin; hexamethylenebisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;idramantone; ilmofosine; ilomastat; imatinib (Gleevec®), imiquimod;immunostimulant peptides; insulin-like growth factor-1 receptorinhibitor; interferon agonists; interferons; interleukins; iobenguane;iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole;isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F;lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinansulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocytealpha interferon; leuprolide+estrogen+progesterone; leuprorelin;levamisole; liarozole; linear polyamine analogue; lipophilicdisaccharide peptide; lipophilic platinum compounds; lissoclinamide 7;lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone;loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lyticpeptides; maitansine; mannostatin A; marimastat; masoprocol; maspin;matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril;merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor;mifepristone; miltefosine; mirimostim; mitoguazone; mitolactol;mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; Erbitux, humanchorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wallsk; mopidamol; mustard anticancer agent; mycaperoxide B; mycobacterialcell wall extract; myriaporone; N-acetyldinaline; N-substitutedbenzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin;naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid;nilutamide; nisamycin: nitric oxide modulators; nitroxide antioxidant;nitrullyn; oblimersen (Genasense®); O6-benzylguanine; octreotide;okicenone; oligonucleotides; onapristone; ondansetron; ondansetron;oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin;oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel derivatives;palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene;parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfatesodium; pentostatin; pentrozole; perflubron; perfosfamide; perillylalcohol; phenazinomycin; phenylacetate; phosphatase inhibitors;picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetinA; placetin B; plasminogen activator inhibitor; platinum complex;platinum compounds; platinum-triamine complex; porfimer sodium;porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2;proteasome inhibitors; protein A-based immune modulator; protein kinaseC inhibitor; protein kinase C inhibitors, microalgal; protein tyrosinephosphatase inhibitors; purine nucleoside phosphorylase inhibitors;purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethyleneconjugate; raf antagonists; raltitrexed; ramosetron; ras farnesylprotein transferase inhibitors; ras inhibitors; ras-GAP inhibitor;retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;ribozymes; RII retinamide; rohitukine; romurtide; roquinimex; rubiginoneB1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim;Sdi 1 mimetics; semustine; senescence derived inhibitor 1; senseoligonucleotides; signal transduction inhibitors; sizofiran; sobuzoxane;sodium borocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stipiamide; stromelysininhibitors; sulfinosine; superactive vasoactive intestinal peptideantagonist; suradista; suramin; swainsonine; tallimustine; tamoxifenmethiodide; tauromustine; tazarotene; tecogalan sodium; tegafur;tellurapyrylium; telomerase inhibitors; temoporfin; teniposide;tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline;thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietinreceptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyletiopurpurin; tirapazamine; titanocene bichloride; topsentin;toremifene; translation inhibitors; tretinoin; triacetyluridine;triciribine; trimetrexate; triptorelin; tropisetron; turosteride;tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex;urogenital sinus-derived growth inhibitory factor; urokinase receptorantagonists; vapreotide; variolin B; velaresol; veramine; verdins;verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone;zeniplatin; zilascorb; and zinostatin stimalamer.

Specific second active agents include, but are not limited to,2-methoxyestradiol, telomestatin, inducers of apoptosis in multiplemyeloma cells (such as, for example, TRAIL), statins, semaxanib,cyclosporin, etanercept, doxycycline, bortezomib, oblimersen(Genasense®), remicade, docetaxel, celecoxib, melphalan, dexamethasone(Decadron®), steroids, gemcitabine, cisplatinum, temozolomide,etoposide, cyclophosphamide, temodar, carboplatin, procarbazine,gliadel, tamoxifen, topotecan, methotrexate, Arisa®, taxol, taxotere,fluorouracil, leucovorin, irinotecan, xeloda, CPT-11, interferon alpha,pegylated interferon alpha (e.g., PEG INTRON-A), capecitabine,cisplatin, thiotepa, fludarabine, carboplatin, liposomal daunorubicin,cytarabine, doxetaxol, pacilitaxel, vinblastine, IL-2, GM-CSF,dacarbazine, vinorelbine, zoledronic acid, palmitronate, biaxin,busulphan, prednisone, bisphosphonate, arsenic trioxide, vincristine,doxorubicin (Doxil®), paclitaxel, ganciclovir, adriamycin, estramustinesodium phosphate (Emcyt®), sulindac, and etoposide.

In another embodiment, examples of specific second agents according tothe indications to be treated, prevented, or managed can be found in thefollowing references, all of which are incorporated herein in theirentireties: U.S. Pat. Nos. 6,281,230 and 5,635,517; U.S. publicationnos. 2004/0220144, 2004/0190609, 2004/0087546, 2005/0203142,2004/0091455, 2005/0100529, 2005/0214328, 2005/0239842, 2006/0154880,2006/0122228, and 2005/0143344; and U.S. provisional application No.60/631,870.

Examples of second active agents that may be used for the treatment,prevention and/or management of pain include, but are not limited to,conventional therapeutics used to treat or prevent pain such asantidepressants, anticonvulsants, antihypertensives, anxiolytics,calcium channel blockers, muscle relaxants, non-narcotic analgesics,opioid analgesics, anti-inflammatories, cox-2 inhibitors,immunomodulatory agents, alpha-adrenergic receptor agonists orantagonists, immunosuppressive agents, corticosteroids, hyperbaricoxygen, ketamine, other anesthetic agents, NMDA antagonists, and othertherapeutics found, for example, in the Physician's Desk Reference 2003.Specific examples include, but are not limited to, salicylic acidacetate (Aspirin®), celecoxib (Celebrex®), Enbrel®, ketamine, gabapentin(Neurontin®), phenyloin (Dilantin®), carbamazepine (Tegretol®),oxcarbazepine (Trileptal®), valproic acid (Depakene®), morphine sulfate,hydromorphone, prednisone, griseofulvin, penthonium, alendronate,dyphenhydramide, guanethidine, ketorolac (Acular®), thyrocalcitonin,dimethylsulfoxide (DMSO®), clonidine (Catapress®), bretylium,ketanserin, reserpine, droperidol, atropine, phentolamine, bupivacaine,lidocaine, acetaminophen, nortriptyline (Pamelor®), amitriptyline(Elavil®), imipramine (Tofranil®), doxepin (Sinequan®), clomipramine(Anafranil®), fluoxetine (Prozac®), sertraline (Zoloft®), naproxen,nefazodone (Serzone®), venlafaxine (Effexor®), trazodone (Desyrel®),bupropion (Wellbutrin®), mexiletine, nifedipine, propranolol, tramadol,lamotrigine, vioxx, ziconotide, ketamine, dextromethorphan,benzodiazepines, baclofen, tizanidine and phenoxybenzamine.

Examples of second active agents that may be used for the treatment,prevention and/or management of macular degeneration and relatedsyndromes include, but are not limited to, a steroid, a lightsensitizer, an integrin, an antioxidant, an interferon, a xanthinederivative, a growth hormone, a neutrotrophic factor, a regulator ofneovascularization, an anti-VEGF antibody, a prostaglandin, anantibiotic, a phytoestrogen, an anti-inflammatory compound or anantiangiogenesis compound, or a combination thereof. Specific examplesinclude, but are not limited to, verteporfin, purlytin, an angiostaticsteroid, rhuFab, interferon-2α, pentoxifylline, tin etiopurpurin,motexafin, lucentis, lutetium,9-fluoro-11,21-dihydroxy-16,17-1-methylethylidinebis(oxy)pregna-1,4-diene-3,20-dione,latanoprost (see U.S. Pat. No. 6,225,348), tetracycline and itsderivatives, rifamycin and its derivatives, macrolides, metronidazole(U.S. Pat. Nos. 6,218,369 and 6,015,803), genistein, genistin, 6′-O-Malgenistin, 6′-O-Ac genistin, daidzein, daidzin, 6′-O-Mal daidzin, 6′-O-Acdaidzin, glycitein, glycitin, 6′-O-Mal glycitin, biochanin A,formononetin (U.S. Pat. No. 6,001,368), triamcinolone acetomide,dexamethasone (U.S. Pat. No. 5,770,589), thalidomide, glutathione (U.S.Pat. No. 5,632,984), basic fibroblast growth factor (bFGF), transforminggrowth factor b (TGF-b), brain-derived neurotrophic factor (BDNF),plasminogen activator factor type 2 (PAI-2), EYE 101 (EyetechPharmaceuticals), LY333531 (Eli Lilly), Miravant, and RETISERT implant(Bausch & Lomb). All of the references cited herein are incorporated intheir entireties by reference.

Examples of second active agents that may be used for the treatment,prevention and/or management of skin diseases include, but are notlimited to, keratolytics, retinoids, α-hydroxy acids, antibiotics,collagen, botulinum toxin, interferon, steroids. and immunomodulatoryagents. Specific examples include, but are not limited to,5-fluorouracil, masoprocol, trichloroacetic acid, salicylic acid, lacticacid, ammonium lactate, urea, tretinoin, isotretinoin, antibiotics,collagen, botulinum toxin, interferon, corticosteroid, transretinoicacid and collagens such as human placental collagen, animal placentalcollagen, Dermalogen, AlloDerm, Fascia, Cymetra, Autologen, Zyderm,Zyplast, Resoplast, and Isolagen.

Examples of second active agents that may be used for the treatment,prevention and/or management of pulmonary hypertension and relateddisorders include, but are not limited to, anticoagulants, diuretics,cardiac glycosides, calcium channel blockers, vasodilators, prostacyclinanalogues, endothelin antagonists, phosphodiesterase inhibitors (e.g.,PDE V inhibitors), endopeptidase inhibitors, lipid lowering agents,thromboxane inhibitors, and other therapeutics known to reduce pulmonaryartery pressure. Specific examples include, but are not limited to,warfarin (Coumadin®), a diuretic, a cardiac glycoside, digoxin-oxygen,diltiazem, nifedipine, a vasodilator such as prostacyclin (e.g.,prostaglandin I2 (PGI2), epoprostenol (EPO, Floran®), treprostinil(Remodulin®), nitric oxide (NO), bosentan (Tracleer®), amlodipine,epoprostenol (Floran®), treprostinil (Remodulin®), prostacyclin,tadalafil (Clalis®), simvastatin (Zocor®), omapatrilat (Vanlev®),irbesartan (Avapro®), pravastatin (Pravachol®), digoxin, L-arginine,iloprost, betaprost, and sildenafil (Viagra®).

Examples of second active agents that may be used for the treatment,prevention and/or management of asbestos-related disorders include, butare not limited to, anthracycline, platinum, alkylating agent,oblimersen (Genasense®), cisplatinum, cyclophosphamide, temodar,carboplatin, procarbazine, gliadel, tamoxifen, topotecan, methotrexate,taxotere, irinotecan, capecitabine, cisplatin, thiotepa, fludarabine,carboplatin, liposomal daunorubicin, cytarabine, doxetaxol, pacilitaxel,vinblastine, IL-2, GM-CSF, dacarbazine, vinorelbine, zoledronic acid,palmitronate, biaxin, busulphan, prednisone, bisphosphonate, arsenictrioxide, vincristine, doxorubicin (Doxil®), paclitaxel, ganciclovir.adriamycin, bleomycin, hyaluronidase, mitomycin C, mepacrine, thiotepa,tetracycline and gemcitabine.

Examples of second active agents that may be used for the treatment,prevention and/or management of parasitic diseases include, but are notlimited to, chloroquine, quinine, quinidine, pyrimethamine,sulfadiazine, doxycycline, clindamycin, mefloquine, halofantrine,primaquine, hydroxychloroquine, proguanil, atovaquone, azithromycin,suramin, pentamidine, melarsoprol, nifurtimox, benznidazole,amphotericin B, pentavalent antimony compounds (e.g., sodiumstiboglucuronate), interfereon gamma, itraconazole, a combination ofdead promastigotes and BCG, leucovorin, corticosteroids, sulfonamide,spiramycin, IgG (serology), trimethoprim, and sulfamethoxazole.

Examples of second active agents that may be used for the treatment,prevention and/or management of immunodeficiency disorders include, butare not limited to: antibiotics (therapeutic or prophylactic) such as,but not limited to, ampicillin, tetracycline, penicillin,cephalosporins, streptomycin, kanamycin, and erythromycin; antiviralssuch as, but not limited to, amantadine, rimantadine, acyclovir, andribavirin; immunoglobulin; plasma; immunologic enhancing drugs such as,but not limited to, levami sole and isoprinosine; biologics such as, butnot limited to, gammaglobulin, transfer factor, interleukins, andinterferons; hormones such as, but not limited to, thymic; and otherimmunologic agents such as, but not limited to, B cell stimulators(e.g., BAFF/BlyS), cytokines (e.g., IL-2, IL-4, and IL-5), growthfactors (e.g., TGF-α), antibodies (e.g., anti-CD40 and IgM),oligonucleotides containing unmethylated CpG motifs, and vaccines (e.g.,viral and tumor peptide vaccines).

Examples of second active agents that may be used for the treatment,prevention and/or management of CNS disorders include, but are notlimited to: opioids; a dopamine agonist or antagonist, such as, but notlimited to, Levodopa, L-DOPA, cocaine, α-methyl-tyrosine, reserpine,tetrabenazine, benzotropine, pargyline, fenodolpam mesylate,cabergoline, pramipexole dihydrochloride, ropinorole, amantadinehydrochloride, selegiline hydrochloride, carbidopa, pergolide mesylate,Sinemet CR, and Symmetrel; a MAO inhibitor, such as, but not limited to,iproniazid, clorgyline, phenelzine and isocarboxazid; a COMT inhibitor,such as, but not limited to, tolcapone and entacapone; a cholinesteraseinhibitor, such as, but not limited to, physostigmine saliclate,physostigmine sulfate, physostigmine bromide, meostigmine bromide,neostigmine methylsulfate, ambenonim chloride, edrophonium chloride,tacrine, pralidoxime chloride, obidoxime chloride, trimedoxime bromide,diacetyl monoxim, endrophonium, pyridostigmine, and demecarium; ananti-inflammatory agent, such as, but not limited to, naproxen sodium,diclofenac sodium, diclofenac potassium, celecoxib, sulindac, oxaprozin,diflunisal, etodolac, meloxicam, ibuprofen, ketoprofen, nabumetone,refecoxib, methotrexate, leflunomide, sulfasalazine, gold salts, Rho-DImmune Globulin, mycophenylate mofetil, cyclosporine, azathioprine,tacrolimus, basiliximab, daclizumab, salicylic acid, acetylsalicylicacid, methyl salicylate, diflunisal, salsalate, olsalazine,sulfasalazine, acetaminophen, indomethacin, sulindac, mefenamic acid,meclofenamate sodium, tolmetin, ketorolac, dichlofenac, flurbinprofen,oxaprozin, piroxicam, meloxicam, ampiroxicam, droxicam, pivoxicam,tenoxicam, phenylbutazone, oxyphenbutazone, antipyrine, aminopyrine,apazone, zileuton, aurothioglucose, gold sodium thiomalate, auranofin,methotrexate, colchicine, allopurinol, probenecid, sulfinpyrazone andbenzbromarone or betamethasone and other glucocorticoids; and anantiemetic agent, such as, but not limited to, metoclopromide,domperidone, prochlorperazine, promethazine, chlorpromazine,trimethobenzamide, ondansetron, granisetron, hydroxyzine, acetylleucinemonoethanolamine, alizapride, azasetron, benzquinamide, bietanautine,bromopride, buclizine, clebopride, cyclizine, dimenhydrinate,diphenidol, dolasetron, meclizine, methallatal, metopimazine, nabilone,oxyperndyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinol,thiethylperazine, thioproperazine, tropisetron, and a mixture thereof.

Examples of second active agents that may be used for the treatment,prevention and/or management of CNS injuries and related syndromesinclude, but are not limited to, immunomodulatory agents,immunosuppressive agents, antihypertensives, anticonvulsants,fibrinolytic agents, antiplatelet agents, antipsychotics,antidepressants, benzodiazepines, buspirone, amantadine, and other knownor conventional agents used in patients with CNS injury/damage andrelated syndromes. Specific examples include, but are not limited to:steroids (e.g., glucocorticoids, such as, but not limited to,methylprednisolone, dexamethasone and betamethasone); ananti-inflammatory agent, including, but not limited to, naproxen sodium,diclofenac sodium, diclofenac potassium, celecoxib, sulindac, oxaprozin,diflunisal, etodolac, meloxicam, ibuprofen, ketoprofen, nabumetone,refecoxib, methotrexate, leflunomide, sulfasalazine, gold salts, RHo-DImmune Globulin, mycophenylate mofetil, cyclosporine, azathioprine,tacrolimus, basiliximab, daclizumab, salicylic acid, acetylsalicylicacid, methyl salicylate, diflunisal, salsalate, olsalazine,sulfasalazine, acetaminophen, indomethacin, sulindac, mefenamic acid,meclofenamate sodium, tolmetin, ketorolac, dichlofenac, flurbinprofen,oxaprozin, piroxicam, meloxicam. ampiroxicam, droxicam, pivoxicam,tenoxicam, phenylbutazone, oxyphenbutazone, antipyrine, aminopyrine,apazone, zileuton, aurothioglucose, gold sodium thiomalate, auranofin,methotrexate, colchicine, allopurinol, probenecid, sulfinpyrazone andbenzbromarone; a cAMP analog including, but not limited to, db-cAMP; anagent comprising a methylphenidate drug, which comprisesl-threo-methylphenidate, d-threo-methylphenidate,dl-threo-methylphenidate, l-erythro-methylphenidate,d-erythro-methylphenidate, dl-erythro-methylphenidate, and a mixturethereof; and a diuretic agent such as, but not limited to, mannitol,furosemide, glycerol, and urea.

Examples of second active agent that may be used for the treatment,prevention and/or management of dysfunctional sleep and relatedsyndromes include, but are not limited to, a tricyclic antidepressantagent, a selective serotonin reuptake inhibitor, an antiepileptic agent(gabapentin, pregabalin, carbamazepine, oxcarbazepine, levitiracetam,topiramate), an antiaryhthmic agent, a sodium channel blocking agent, aselective inflammatory mediator inhibitor, an opioid agent, a secondimmunomodulatory compound, a combination agent, and other known orconventional agents used in sleep therapy. Specific examples include,but are not limited to, Neurontin, oxycontin, morphine, topiramate,amitryptiline, nortryptiline, carbamazepine, Levodopa, L-DOPA, cocaine,α-methyl-tyrosine, reserpine, tetrabenazine, benzotropine, pargyline,fenodolpam mesylate, cabergoline, pramipexole dihydrochloride,ropinorole, amantadine hydrochloride, selegiline hydrochloride,carbidopa, pergolide mesylate, Sinemet CR, Symmetrel, iproniazid,clorgyline, phenelzine, isocarboxazid, tolcapone, entacapone,physostigmine saliclate, physostigmine sulfate, physostigmine bromide,meostigmine bromide, neostigmine methylsulfate, ambenonim chloride,edrophonium chloride, tacrine, pralidoxime chloride, obidoxime chloride,trimedoxime bromide, diacetyl monoxim, endrophonium, pyridostigmine,demecarium, naproxen sodium, diclofenac sodium, diclofenac potassium,celecoxib, sulindac, oxaprozin, diflunisal, etodolac, meloxicam,ibuprofen, ketoprofen, nabumetone, refecoxib, methotrexate, leflunomide,sulfasalazine, gold salts, RHo-D Immune Globulin, mycophenylate mofetil,cyclosporine, azathioprine, tacrolimus, basiliximab, daclizumab,salicylic acid, acetylsalicylic acid, methyl salicylate, diflunisal,salsalate, olsalazine, sulfasalazine, acetaminophen, indomethacin,sulindac, mefenamic acid, meclofenamate sodium, tolmetin, ketorolac,dichlofenac, flurbinprofen, oxaprozin, piroxicam, meloxicam,ampiroxicam, droxicam, pivoxicam, tenoxicam, phenylbutazone,oxyphenbutazone, antipyrine, aminopyrine, apazone, zileuton,aurothioglucose, gold sodium thiomalate, auranofin, methotrexate,colchicine, allopurinol, probenecid, sulfinpyrazone, benzbromarone,betamethasone and other glucocorticoids, metoclopromide, domperidone,prochlorperazine, promethazine, chlorpromazine, trimethobenzamide,ondansetron, granisetron, hydroxyzine, acetylleucine monoethanolamine,alizapride, azasetron, benzquinamide, bietanautine, bromopride,buclizine, clebopride, cyclizine, dimenhydrinate, diphenidol,dolasetron, meclizine, methallatal, metopimazine, nabilone, oxyperndyl,pipamazine, scopolamine, sulpiride, tetrahydrocannabinol,thiethylperazine, thioproperazine, tropisetron, and a mixture thereof.

Examples of second active agents that may be used for the treatment,prevention and/or management of hemoglobinopathy and related disordersinclude, but are not limited to: interleukins, such as IL-2 (includingrecombinant IL-II (“rIL2”) and canarypox IL-2), IL-10, IL-12, and IL-18;interferons, such as interferon alfa-2a, interferon alfa-2b, interferonalfa-n1, interferon alfa-n3, interferon beta-I a, and interferon gamma-Ib; and G-CSF; hydroxyurea; butyrates or butyrate derivatives; nitrousoxide; hydroxy urea; HEMOXIN™ (NIPRISAN™; see U.S. Pat. No. 5,800,819);Gardos channel antagonists such as clotrimazole and triaryl methanederivatives; Deferoxamine; protein C; and transfusions of blood, or of ablood substitute such as Hemospan™ or Hemospan™ PS (Sangart).

Administration of a compound provided herein, or a pharmaceuticallyacceptable salt, solvate, clathrate, stereoisomer or prodrug thereof,and the second active agents to a patient can occur simultaneously orsequentially by the same or different routes of administration. Thesuitability of a particular route of administration employed for aparticular active agent will depend on the active agent itself (e.g.,whether it can be administered orally without decomposing prior toentering the blood stream) and the disease being treated. One ofadministration for compounds provided herein is oral. Routes ofadministration for the second active agents or ingredients are known tothose of ordinary skill in the art. See, e.g., Physicians' DeskReference (60^(th) ed., 2006).

In one embodiment, the second active agent is administered intravenouslyor subcutaneously and once or twice daily in an amount of from about 1to about 1000 mg, from about 5 to about 500 mg, from about 10 to about350 mg, or from about 50 to about 200 mg. The specific amount of thesecond active agent will depend on the specific agent used, the type ofdisease being treated or managed, the severity and stage of disease, andthe amount(s) of compounds provided herein and any optional additionalactive agents concurrently administered to the patient.

As discussed elsewhere herein, also encompassed is a method of reducing,treating and/or preventing adverse or undesired effects associated withconventional therapy including, but not limited to, surgery,chemotherapy, radiation therapy, hormonal therapy, biological therapyand immunotherapy. Compounds provided herein and other activeingredients can be administered to a patient prior to, during, or afterthe occurrence of the adverse effect associated with conventionaltherapy.

4.4 Cycling Therapy

In certain embodiments, the prophylactic or therapeutic agents providedherein are cyclically administered to a patient. Cycling therapyinvolves the administration of an active agent for a period of time,followed by a rest (i.e., discontinuation of the administration) for aperiod of time, and repeating this sequential administration. Cyclingtherapy can reduce the development of resistance to one or more of thetherapies, avoid or reduce the side effects of one of the therapies,and/or improve the efficacy of the treatment.

Consequently. in one embodiment, a compound provided herein isadministered daily in a single or divided doses in a four to six weekcycle with a rest period of about a week or two weeks. Cycling therapyfurther allows the frequency, number, and length of dosing cycles to beincreased. Thus, another embodiment encompasses the administration of acompound provided herein for more cycles than are typical when it isadministered alone. In yet another embodiment, a compound providedherein is administered for a greater number of cycles than wouldtypically cause dose-limiting toxicity in a patient to whom a secondactive ingredient is not also being administered.

In one embodiment, a compound provided herein is administered daily andcontinuously for three or four weeks at a dose of from about 0.1 mg toabout 500 mg per day, followed by a rest of one or two weeks. In otherembodiments, the dose can be from about 1 mg to about 300 mg, from about0.1 mg to about 150 mg, from about 1 mg to about 200 mg, from about 10mg to about 100 mg, from about 0.1 mg to about 50 mg, from about 1 mg toabout 50 mg, from about 10 mg to about 50 mg, from about 20 mg to about30 mg, or from about 1 mg to about 20 mg, followed by a rest.

In one embodiment, a compound provided herein and a second activeingredient are administered orally, with administration of the compoundprovided herein occurring 30 to 60 minutes prior to the second activeingredient, during a cycle of four to six weeks. In another embodiment,the combination of a compound provided herein and a second activeingredient is administered by intravenous infusion over about 90 minutesevery cycle.

Typically, the number of cycles during which the combination treatmentis administered to a patient will be from about one to about 24 cycles,from about two to about 16 cycles, or from about four to about threecycles.

4.5 Pharmaceutical Compositions and Dosage Forms

Pharmaceutical compositions can be used in the preparation ofindividual, single unit dosage forms. Pharmaceutical compositions anddosage forms provided herein comprise a compound provided herein, or apharmaceutically acceptable salt, solvate, stereoisomer, clathrate, orprodrug thereof. Pharmaceutical compositions and dosage forms canfurther comprise one or more excipients.

Pharmaceutical compositions and dosage forms provided herein can alsocomprise one or more additional active ingredients. Examples of optionalsecond, or additional, active ingredients are disclosed in Section 4.3,above.

Single unit dosage forms provided herein are suitable for oral, mucosal(e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g.,subcutaneous, intravenous, bolus injection, intramuscular, orintraarterial), topical (e.g., eye drops or other ophthalmicpreparations), transdermal or transcutaneous administration to apatient. Examples of dosage forms include, but are not limited to:tablets; caplets; capsules, such as soft elastic gelatin capsules;cachets; troches; lozenges; dispersions; suppositories; powders;aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage formssuitable for oral or mucosal administration to a patient, includingsuspensions (e.g., aqueous or non-aqueous liquid suspensions,oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a patient; eye drops or other ophthalmic preparations suitable fortopical administration; and sterile solids (e.g., crystalline oramorphous solids) that can be reconstituted to provide liquid dosageforms suitable for parenteral administration to a patient.

The composition, shape, and type of dosage forms will typically varydepending on their use. For example, a dosage form used in the acutetreatment of a disease may contain larger amounts of one or more of theactive ingredients it comprises than a dosage form used in the chronictreatment of the same disease. Similarly, a parenteral dosage form maycontain smaller amounts of one or more of the active ingredients itcomprises than an oral dosage form used to treat the same disease. Theseand other ways in which specific dosage forms are used will vary fromone another will be readily apparent to those skilled in the art. See,e.g., Remington's Pharmaceutical Sciences, 20^(th) ed., Mack Publishing,Easton Pa. (2000).

In one embodiment, pharmaceutical compositions and dosage forms compriseone or more excipients. Suitable excipients are well known to thoseskilled in the art of pharmacy, and non-limiting examples of suitableexcipients are provided herein. Whether a particular excipient issuitable for incorporation into a pharmaceutical composition or dosageform depends on a variety of factors well known in the art including,but not limited to, the way in which the dosage form will beadministered to a patient. For example, oral dosage forms such astablets may contain excipients not suited for use in parenteral dosageforms. The suitability of a particular excipient may also depend on thespecific active ingredients in the dosage form. For example, thedecomposition of some active ingredients may be accelerated by someexcipients such as lactose, or when exposed to water. Active ingredientsthat comprise primary or secondary amines are particularly susceptibleto such accelerated decomposition. Consequently, provided arepharmaceutical compositions and dosage forms that contain little, ifany, lactose other mono- or di-saccharides. As used herein, the term“lactose-free” means that the amount of lactose present, if any, isinsufficient to substantially increase the degradation rate of an activeingredient.

Lactose-free compositions can comprise excipients that are well known inthe art and are listed, for example, in the U.S. Pharmacopeia (USP)25-NF20 (2002). In general, lactose-free compositions comprise activeingredients, a binder/filler, and a lubricant in pharmaceuticallycompatible and pharmaceutically acceptable amounts. In one embodiment,lactose-free dosage forms comprise active ingredients, microcrystallinecellulose, pre-gelatinized starch, and magnesium stearate.

Also provided are anhydrous pharmaceutical compositions and dosage formscomprising active ingredients, since water can facilitate thedegradation of some compounds. For example, the addition of water (e.g.,5%) is widely accepted in the pharmaceutical arts as a means ofsimulating long-term storage in order to determine characteristics suchas shelf-life or the stability of formulations over time. See, e.g.,Jens T. Carstensen, Drug Stability: Principles & Practice, 2d. Ed.,Marcel Dekker, NY, N.Y., 1995, pp. 379-80. In effect, water and heataccelerate the decomposition of some compounds. Thus, the effect ofwater on a formulation can be of great significance since moistureand/or humidity are commonly encountered during manufacture, handling,packaging, storage, shipment, and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms can be preparedusing anhydrous or low moisture containing ingredients and low moistureor low humidity conditions. Pharmaceutical compositions and dosage formsthat comprise lactose and at least one active ingredient that comprisesa primary or secondary amine are anhydrous if substantial contact withmoisture and/or humidity during manufacturing, packaging, and/or storageis expected.

An anhydrous pharmaceutical composition should be prepared and storedsuch that its anhydrous nature is maintained. Accordingly, anhydrouscompositions are, in one embodiment, packaged using materials known toprevent exposure to water such that they can be included in suitableformulary kits. Examples of suitable packaging include, but are notlimited to, hermetically sealed foils, plastics, unit dose containers(e.g., vials), blister packs, and strip packs.

Also provided are pharmaceutical compositions and dosage forms thatcomprise one or more compounds that reduce the rate by which an activeingredient will decompose. Such compounds, which are referred to hereinas “stabilizers,” include, but are not limited to, antioxidants such asascorbic acid, pH buffers, or salt buffers.

Like the amounts and types of excipients, the amounts and specific typesof active ingredients in a dosage form may differ depending on factorssuch as, but not limited to, the route by which it is to be administeredto patients. In one embodiment, dosage forms comprise a compoundprovided herein in an amount of from about 0.10 to about 500 mg. Inother embodiments, dosage forms comprise a compound provided herein inan amount of about 0.1, 1, 2, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, 50,100, 150, 200, 250, 300, 350, 400, 450, or 500 mg.

In other embodiments, dosage forms comprise the second active ingredientin an amount of 1 to about 1000 mg, from about 5 to about 500 mg, fromabout 10 to about 350 mg, or from about 50 to about 200 mg. Of course,the specific amount of the second active agent will depend on thespecific agent used, the diseases or disorders being treated or managed,and the amount(s) of a compound provided herein, and any optionaladditional active agents concurrently administered to the patient.

4.5.1 Oral Dosage Forms

Pharmaceutical compositions that are suitable for oral administrationcan be provided as discrete dosage forms, such as, but not limited to,tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g.,flavored syrups). Such dosage forms contain predetermined amounts ofactive ingredients, and may be prepared by methods of pharmacy wellknown to those skilled in the art, See generally, Remington'sPharmaceutical Sciences, 20th ed., Mack Publishing, Easton Pa. (2000).

Oral dosage forms provided herein are prepared by combining the activeingredients in an intimate admixture with at least one excipientaccording to conventional pharmaceutical compounding techniques.Excipients can take a wide variety of forms depending on the form ofpreparation desired for administration. For example, excipients suitablefor use in oral liquid or aerosol dosage forms include, but are notlimited to, water, glycols, oils, alcohols, flavoring agents,preservatives, and coloring agents. Examples of excipients suitable foruse in solid oral dosage forms (e.g., powders, tablets, capsules, andcaplets) include, but are not limited to, starches, sugars,micro-crystalline cellulose, diluents, granulating agents, lubricants,binders, and disintegrating agents.

In one embodiment, oral dosage forms are tablets or capsules, in whichcase solid excipients are employed. In another embodiment, tablets canbe coated by standard aqueous or nonaqueous techniques. Such dosageforms can be prepared by any of the methods of pharmacy. In general,pharmaceutical compositions and dosage forms are prepared by uniformlyand intimately admixing the active ingredients with liquid carriers,finely divided solid carriers, or both, and then shaping the productinto the desired presentation if necessary.

For example, a tablet can be prepared by compression or molding.Compressed tablets can be prepared by compressing in a suitable machinethe active ingredients in a free-flowing form such as powder orgranules, optionally mixed with an excipient. Molded tablets can be madeby molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

Examples of excipients that can be used in oral dosage forms providedherein include, but are not limited to, binders, fillers, disintegrants,and lubricants. Binders suitable for use in pharmaceutical compositionsand dosage forms include, but are not limited to, corn starch, potatostarch, or other starches, gelatin, natural and synthetic gums such asacacia, sodium alginate, alginic acid, other alginates, powderedtragacanth, guar gum, cellulose and its derivatives (e.g., ethylcellulose, cellulose acetate, carboxymethyl cellulose calcium, sodiumcarboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose,pre-gelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos.2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.

Suitable forms of microcrystalline cellulose include, but are notlimited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICELRC-581, AVICEL-PH-105 (available from FMC Corporation, American ViscoseDivision, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof. Anspecific binder is a mixture of microcrystalline cellulose and sodiumcarboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or lowmoisture excipients or additives include AVICEL-PH-103™ and Starch 1500LM.

Examples of fillers suitable for use in the pharmaceutical compositionsand dosage forms provided herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.The binder or filler in pharmaceutical compositions is, in oneembodiment, present in from about 50 to about 99 weight percent of thepharmaceutical composition or dosage form.

Disintegrants may be used in the compositions to provide tablets thatdisintegrate when exposed to an aqueous environment. Tablets thatcontain too much disintegrant may disintegrate in storage, while thosethat contain too little may not disintegrate at a desired rate or underthe desired conditions. Thus, a sufficient amount of disintegrant thatis neither too much nor too little to detrimentally alter the release ofthe active ingredients may be used to form solid oral dosage forms. Theamount of disintegrant used varies based upon the type of formulation,and is readily discernible to those of ordinary skill in the art. In oneembodiment, pharmaceutical compositions comprise from about 0.5 to about15 weight percent of disintegrant, or from about 1 to about 5 weightpercent of disintegrant.

Disintegrants that can be used in pharmaceutical compositions and dosageforms include, but are not limited to, agar-agar, alginic acid, calciumcarbonate, microcrystalline cellulose, croscarmellose sodium,crospovidone, polacrilin potassium, sodium starch glycolate, potato ortapioca starch, other starches, pre-gelatinized starch, other starches,clays, other algins, other celluloses, gums, and mixtures thereof.

Lubricants that can be used in pharmaceutical compositions and dosageforms include, but are not limited to, calcium stearate, magnesiumstearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol,polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate,talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil,sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zincstearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof.Additional lubricants include, for example, a syloid silica gel(AEROSIL200, manufactured by W.R. Grace Co. of Baltimore, Md.), acoagulated aerosol of synthetic silica (marketed by Degussa Co. ofPlano, Tex.), CAB-O-SIL (a pyrogenic silicon dioxide product sold byCabot Co. of Boston, Mass.), and mixtures thereof. If used at all,lubricants may be used in an amount of less than about 1 weight percentof the pharmaceutical compositions or dosage forms into which they areincorporated.

In one embodiment, a solid oral dosage form comprises a compoundprovided herein, anhydrous lactose, microcrystalline cellulose,polyvinylpyrrolidone, stearic acid, colloidal anhydrous silica, andgelatin.

4.5.2 Controlled Release Dosage Forms

Active ingredients such as the compounds provided herein can beadministered by controlled release means or by delivery devices that arewell known to those of ordinary skill in the art. Examples include, butare not limited to, those described in U.S. Pat. Nos. 3,845,770;3,916,899; 3,536,809; 3,598,123; and 4,008,719; 5,674,533; 5,059,595;5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,639,480;5,733,566; 5,739,108; 5,891,474; 5,922,356; 5,972,891; 5,980,945;5,993,855; 6,045,830; 6,087,324; 6,113,943; 6,197,350; 6,248,363;6,264,970; 6,267,981; 6,376,461; 6,419,961; 6,589,548; 6,613,358;6,699,500 each of which is incorporated herein by reference. Such dosageforms can be used to provide slow or controlled release of one or moreactive ingredients using, for example, hydropropylmethyl cellulose,other polymer matrices, gels, permeable membranes, osmotic systems,multilayer coatings, microparticles, liposomes, microspheres, or acombination thereof to provide the desired release profile in varyingproportions. Suitable controlled release formulations known to those ofordinary skill in the art, including those described herein, can bereadily selected for use with the active ingredients provided herein.Thus, the compositions provided encompasse single unit dosage formssuitable for oral administration such as, but not limited to, tablets,capsules, gelcaps, and caplets that are adapted for controlled release.

All controlled release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non controlledcounterparts. Ideally, the use of an optimally designed controlledrelease preparation in medical treatment is characterized by a minimumof drug substance being employed to cure or control the condition in aminimum amount of time. Advantages of controlled release formulationsinclude extended activity of the drug, reduced dosage frequency, andincreased subject compliance. In addition, controlled releaseformulations can be used to affect the time of onset of action or othercharacteristics, such as blood levels of the drug, and can thus affectthe occurrence of side (e.g., adverse) effects.

Most controlled release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release of otheramounts of drug to maintain this level of therapeutic or prophylacticeffect over an extended period of time. In order to maintain thisconstant level of drug in the body, the drug must be released from thedosage form at a rate that will replace the amount of drug beingmetabolized and excreted from the body. Controlled release of an activeingredient can be stimulated by various conditions including, but notlimited to, pH, temperature, enzymes, water, or other physiologicalconditions or compounds.

In certain embodiments, the drug may be administered using intravenousinfusion, an implantable osmotic pump, a transdermal patch, liposomes,or other modes of administration. In one embodiment, a pump may be used(see, Sefton, CRC Crit. Ref Biomed. Eng. 14:201 (1987); Buchwald et al.,Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).In another embodiment, polymeric materials can be used. In yet anotherembodiment, a controlled release system can be placed in a subject at anappropriate site determined by a practitioner of skill, i.e., thusrequiring only a fraction of the systemic dose (see, e.g., Goodson,Medical Applications of Controlled Release, vol. 2, pp. 115-138 (1984)).Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)). The active ingredient can be dispersedin a solid inner matrix, e.g., polymethylmethacrylate,polybutylmethacrylate, plasticized or unplasticized polyvinylchloride,plasticized nylon, plasticized polyethyleneterephthalate, naturalrubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene,ethylene-vinylacetate copolymers, silicone rubbers,polydimethylsiloxanes, silicone carbonate copolymers, hydrophilicpolymers such as hydrogels of esters of acrylic and methacrylic acid,collagen, cross-linked polyvinylalcohol and cross-linked partiallyhydrolyzed polyvinyl acetate, that is surrounded by an outer polymericmembrane, e.g., polyethylene, polypropylene, ethylene/propylenecopolymers, ethylene/ethyl acrylate copolymers, ethylene/vinylacetatecopolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber,chlorinated polyethylene, polyvinylchloride, vinylchloride copolymerswith vinyl acetate, vinylidene chloride, ethylene and propylene, ionomerpolyethylene terephthalate, butyl rubber epichlorohydrin rubbers,ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcoholterpolymer, and ethylene/vinyloxyethanol copolymer, that is insoluble inbody fluids. The active ingredient then diffuses through the outerpolymeric membrane in a release rate controlling step. The percentage ofactive ingredient in such parenteral compositions is highly dependent onthe specific nature thereof, as well as the needs of the subject.

4.5.3 Parenteral Dosage Forms

Parenteral dosage forms can be administered to patients by variousroutes including, but not limited to, subcutaneous, intravenous(including bolus injection), intramuscular, and intraarterial. In someembodiments, administration of a parenteral dosage form bypassespatients' natural defenses against contaminants, and thus, in theseembodiments, parenteral dosage forms are sterile or capable of beingsterilized prior to administration to a patient. Examples of parenteraldosage forms include, but are not limited to, solutions ready forinjection, dry products ready to be dissolved or suspended in apharmaceutically acceptable vehicle for injection, suspensions ready forinjection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage formsare well known to those skilled in the art. Examples include, but arenot limited to: Water for Injection USP; aqueous vehicles such as, butnot limited to, Sodium Chloride Injection, Ringer's Injection, DextroseInjection, Dextrose and Sodium Chloride Injection, and Lactated Ringer'sInjection; water-miscible vehicles such as, but not limited to, ethylalcohol, polyethylene glycol, and polypropylene glycol; and non-aqueousvehicles such as, but not limited to, corn oil, cottonseed oil, peanutoil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Compounds that increase the solubility of one or more of the activeingredients disclosed herein can also be incorporated into theparenteral dosage forms. For example, cyclodextrin and its derivativescan be used to increase the solubility of a compound provided herein.See, e.g., U.S. Pat. No. 5,134,127, which is incorporated herein byreference.

4.5.4 Topical and Mucosal Dosage Forms

Topical and mucosal dosage forms provided herein include, but are notlimited to, sprays, aerosols, solutions, emulsions, suspensions, eyedrops or other ophthalmic preparations, or other forms known to one ofskill in the art. See, e.g., Remington's Pharmaceutical Sciences,16^(th), 18^(th) and 20^(th) eds., Mack Publishing, Easton Pa. (1980,1990 and 2000); and Introduction to Pharmaceutical Dosage Forms, 4thed., Lea & Febiger, Philadelphia (1985). Dosage forms suitable fortreating mucosal tissues within the oral cavity can be formulated asmouthwashes or as oral gels.

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide topical and mucosal dosage forms encompassedherein are well known to those skilled in the pharmaceutical arts, anddepend on the particular tissue to which a given pharmaceuticalcomposition or dosage form will be applied. In one embodiment,excipients include, but are not limited to, water, acetone, ethanol,ethylene glycol, propylene glycol, butane-1,3-diol, isopropyl myristate,isopropyl palmitate, mineral oil, and mixtures thereof to formsolutions, emulsions or gels, which are non-toxic and pharmaceuticallyacceptable. Moisturizers or humectants can also be added topharmaceutical compositions and dosage forms. Examples of additionalingredients are well known in the art. See, e.g., Remington'sPharmaceutical Sciences, 16^(th), 18^(th) and 20^(th) eds., MackPublishing, Easton Pa. (1980, 1990 and 2000).

The pH of a pharmaceutical composition or dosage form may also beadjusted to improve delivery of one or more active ingredients. Also,the polarity of a solvent carrier, its ionic strength, or tonicity canbe adjusted to improve delivery. Compounds such as stearates can also beadded to pharmaceutical compositions or dosage forms to alter thehydrophilicity or lipophilicity of one or more active ingredients so asto improve delivery. In other embodiments, stearates can serve as alipid vehicle for the formulation, as an emulsifying agent orsurfactant, or as a delivery-enhancing or penetration-enhancing agent.In other embodiments, salts, solvates, prodrugs, clathrates, orstereoisomers of the active ingredients can be used to further adjustthe properties of the resulting composition.

4.6 Kits

In one embodiment, active ingredients provided herein are notadministered to a patient at the same time or by the same route ofadministration. In another embodiment, provided are kits which cansimplify the administration of appropriate amounts of activeingredients.

In one embodiment, a kit comprises a dosage form of a compound providedherein. Kits can further comprise additional active ingredients such asoblimersen (Genasense®), melphalan, G-CSF, GM-CSF, EPO, topotecan,dacarbazine, irinotecan, taxotere, IFN, COX-2 inhibitor, pentoxifylline,ciprofloxacin, dexamethasone, IL2, IL8, IL 18, Ara-C, vinorelbine,isotretinoin, 13 cis-retinoic acid, or a pharmacologically active mutantor derivative thereof, or a combination thereof. Examples of theadditional active ingredients include, but are not limited to, thosedisclosed herein (see, e.g., section 4.3).

In other embodiments, kits can further comprise devices that are used toadminister the active ingredients. Examples of such devices include, butare not limited to, syringes, drip bags, patches, and inhalers.

Kits can further comprise cells or blood for transplantation as well aspharmaceutically acceptable vehicles that can be used to administer oneor more active ingredients. For example, if an active ingredient isprovided in a solid form that must be reconstituted for parenteraladministration, the kit can comprise a sealed container of a suitablevehicle in which the active ingredient can be dissolved to form aparticulate-free sterile solution that is suitable for parenteraladministration. Examples of pharmaceutically acceptable vehiclesinclude, but are not limited to: Water for Injection USP; aqueousvehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water-miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

5. EXAMPLES

Certain embodiments of the claimed subject matter are illustrated by thefollowing non-limiting examples.

Example 12-(2,6-Dioxo-piperidin-3-yl)-4-(2-methoxy-phenoxy)-isoindole-1,3-dione

Step 1:

Methyl iodide (30.2 g, 213 mmol) was added to a stirred mixture of3-nitrophthalic acid (15.0 g, 71.0 mmol) and sodium bicarbonate (23.9 g,284 mmol) in DMF (150 mL) at room temperature, and the mixture was thenheated in an oil bath set to 60° C. for 4 h. The mixture was then pouredinto 700 mL of ice water. After the ice melted, the mixture wasextracted with ethyl acetate (3×150 mL) and the organic phases werewashed with water (7×500 mL), dried (MgSO₄) and evaporated, providing16.2 g of 3-nitrophthalic acid dimethyl ester as a pale yellow solid, in95% yield; ¹H NMR (CDCl₃) δ 3.95 (s, 3H), 4.02 (s, 3H), 7.69 (t, J=8.1Hz, 1H), 8.36 (m, 2H).

Step 2:

A mixture of 3:1 ethanol-conc. HCl (200 mL) was cooled to 0° C. and then3-nitrophthalic acid dimethyl ester (15.0 g, 62.8 mmol) was added.Maintaining the cooling, tin (II) chloride (70.8 g, 314 mmol) was addedportionwise, over a period of 15 min. Following completion of theaddition, the cooling bath was removed and stirring proceeded at roomtemperature. After 2 h, the mixture was neutralized by the addition ofsolid sodium bicarbonate, and the resulting mixture was extracted withethyl acetate (3×150 mL) and the combined extracts were washed withwater (5×250 mL), were dried (MgSO₄) and evaporated, providing 11.3 g of3-aminophthalic acid dimethyl ester as a yellow oil, in 86% yield; ¹HNMR (CDCl₃) δ 3.84 (s, 3H), 3.86 (s, 3H), 5.20 (br, 2H), 6.78 (dd, J=8.5Hz, J=1.0 Hz, 1H), 6.90 (dd, 1H, J=7.3 Hz, J=1.0 Hz, 1H), 7.24 (t, J=7.8Hz, 1H).

Step 3:

A solution of 3-aminophthalic acid dimethyl ester (9.5 g, 45.4 mmol) in1:1 water-conc. HCl (300 mL) was cooled to 0° C.; during cooling, aprecicipitate formed. A solution of NaNO₂ (3.5 g, 50.0 mmol) in 10 mLwater was then added slowly, maintaining the temperature between 0-5° C.throughout the addition. Following completion of the addition, themixture was stirred at this temperature for 10 minutes before adding asolution of KI (11.3 g, 68.3 mmol) in 30 mL of 1:1 water-conc. HCl. Thissolution was added all at once, and then the reaction flask wastransferred immediately to an oil bath preheated to 65° C. The mixturestirred with heating for 10 minutes, and was then cooled in an ice bath.The mixture was extracted with CH₂Cl₂ (3×150 mL) and the combinedorganic extracts were washed with water (3×150 mL), were dried (MgSO₄)and evaporated, and the residue was chromatographed using withhexanes-ethyl acetate gradient. The product, which eluted at 17:3hexanes-ethyl acetate, was a light purple solid, and was then trituratedwith hexanes, filtered, and dried to give 9.7 g (67%) of 3-iodophthalicacid dimethyl ester, as a colorless solid; ¹H NMR (CDCl₃) δ 3.90 (s,3H), 3.99 (s, 3H), 7.19 (t, J=7.9 Hz, 1H), 8.02 (d, J=7.9 Hz, 2H).

Step 4:

A mixture of guaiacol (0.77 g, 6.2 mmol), copper (I) bromide (0.89 g,6.2 mmol), and sodium hydride (0.3 g of a 60% dispersion, 7.5 mmol) in100 mL pyridine was heated to reflux and stirred under nitrogen for 15min. 3-Iodophthalic acid dimethyl ester (2.0 g, 6.2 mmol) was added, andthe resulting mixture was stirred at reflux for 20 h. The mixture wascooled to room temperature and quenched by the addition of saturatedNH₄Cl (15 mL). Volatiles were removed under reduced pressure. Theresidue was partitioned between dilute aqueous HCl (100 mL) and ethylacetate (100 mL), and the aqueous phase was extracted with ethyl acetate(100 mL). The combined organic extracts were washed with dilute aqueousHCl (2×100 mL), saturated Na₂CO₃ (2×100 mL), again with dilute aqueousHCl (2×100 mL) and finally with water (100 mL), and were evaporated.Chromatography in hexanes-ethyl acetate gradient provided 0.75 g of3-(2-methoxy-phenoxy)-phthalic acid dimethyl ester, eluting at 25-30%ethyl acetate, in 38% yield; ¹H NMR (CDCl₃) δ 3.79 (s, 3H), 3.91 (s,3H), 3.95 (s, 3H), 6.86-6.91 (m, 1H), 6.93-7.01 (m, 2H), 7.05 (dd, J=7.9Hz, J=1.6 Hz, 1H), 7.13-7.20 (m, 1H), 7.30 (t, J=8.0 Hz, 1H), 7.66-7.69(m, 1H).

Step 5:

A mixture of 3-(2-methoxy-phenoxy)-phthalic acid dimethyl ester (0.75 g,2.4 mmol) and 3N NaOH (50 mL) in ethanol (100 mL) was heated to refluxfor 2 h. The mixture was cooled and the solvent was removed undervacuum. The residue was dissolved in water (100 mL) and washed withCH₂Cl₂ (2×100 mL), acidified (HCl), and extracted with ethyl acetate(3×75 mL). The combined organic extracts were washed with water (3×75mL), dried (MgSO₄), and evaporated, providing 0.53 g of3-(2-methoxy-phenoxy)-phthalic acid, in 78% yield; ¹H NMR (DMSO-d₆) δ3.76 (s, 3H), 6.82 (dd, J=8.4 Hz, J=0.9 Hz, 1H), 6.95-6.98 (m, 2H),7.16-7.23 (m, 2H), 7.39 (t, J=8.0 Hz, 1H), 7.58 (d, J=8.2 Hz, 1H).

Step 6:

A mixture of 3-(2-methoxy-phenoxy)-phthalic acid (0.51 g, 1.8 mmol) andrac-α-aminoglutarimide hydrochloride (0.29 g, 1.8 mmol) in pyridine (10mL) was heated to reflux for 16 h. The mixture was cooled and evaporatedunder vacuum. The residue was dissolved in ethyl acetate (100 mL) andwashed with dilute aqueous HO (2×100 mL) and water (100 mL), and wasevaporated. The residue was chromatographed using a CH₂Cl₂-methanolgradient, eluting the title compound at 95:5 CH₂Cl₂-methanol, 0.59 g, in88% yield; mp 223-225° C.; HPLC, Waters Symmetry C-18, 3.9×150 mm, 5 μm,1 mL/min, 240 nm, 40/60 CH₃CN/0.1% H₃PO₄, 6.06 (99.46%); ¹H NMR(DMSO-d₆) δ 2.06-2.11 (m, 1H), 2.53-2.64 (m, 2H), 2.83-2.90 (m, 1H),3.75 (s, 1H), 5.15 (dd, J=12.4 Hz, J=5.3 Hz, 1H), 6.85 (d, J=8.5 Hz,1H), 7.06 (t, J=7.3 Hz, 1H), 7.21-7.37 (m, 3H), 7.54 (d, J=7.2 Hz, 1H),7.72 (t, J=7.9 Hz, 1H), 11.13 (s, 1H); ¹³C NMR (DMSO-d₆) δ 21.9, 31.0,48.9, 55.7, 113.7, 116.5, 116.7, 120.7, 121.5, 122.3, 127.2, 133.3,137.0, 141.3, 151.2, 154.7, 165.0, 166.6, 170.0, 172.8; Anal. Calcd forC₂₀H₁₆N₂O₆: C, 63.16; H, 4.24; N. 7.37. Found: C, 63.00; H, 4.24; N,7.29.

Example 2 3-(4-hydroxy-1-oxoisoindolin-2-yl)piperidine-2,6-dione

A 250 mL-3N-RBF was charged with3-(4-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione and H₂O (10 vol)and cooled to 0-5° C. NaNO₂ (1.1 eq) and HCl (1.1 eq) were added, andthe mixture was stirred for 30 minutes. The mixture was then heated to75-80° C. for 2 hours. The mixture was cooled to room temperature,filtered and dried in vacuo (18 h, 35-40° C.). The crude product waspurified by prep-HPLC (Conditions: C18 Symmetry Column, 90:10 H₂O: MeCNisocratic, 60 mL/min flow rate, product retention time ˜30 min) to givean off-white solid (240 mg, 2.4%, 99.5 HPLC AP); mp 296.39° C.; HPLC:Hypersil DBS C8 5 m column, 250×4.6 mm, 35° C.; 99:1 to 85:15 GradientCH₃CN/10 mM aq. KH₂PO₄, 1.0 mL/min over 20 minutes; 7.60 min, 99.5% APat 210/240 nm: ¹H-NMR (DMSO-d₆): 10.97 (1H, br s), 10.11 (1H, br s),7.34 (1H, t), 7.17 (1H, d), 7.03 (1H, d), 5.09 (1H, dd), 4.25 (2H, dd),2.97-2.85 (1H, m), 2.62-2.36 (2H, m), 2.03-1.96 (1H, m) ppm; ¹³C-NMR(DMSO-d₆): 172.85, 171.04, 168.27, 152.55, 133.41, 129.44, 127.94,117.97, 113.71, 51.59, 45.09, 31.22, 22.42 ppm; LC-MS ES⁺ (M+1) 261;CHN-Analysis, calcd for C₁₃H₁₂N₂O₄: C, 60.00%; H, 4.65%; N, 10.76%.Found: C, 59.54%; H, 4.88%; N, 10.48%.

Example 3 4-Benzyloxy-2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dione

Step 1:

A mixture of 3-hydroxyphthalic anhydride (4.96 g, 30.2 mmol) in methanol(60 mL) was refluxed for 3 h, cooled to room temperature, and thesolvent was evaporated under vacuum. The residue and sodium bicarbonate(7.11 g, 84.6 mmol) were suspended in DMF (40 mL). Iodomethane (4.53 mL,72.5 mmol) was added and the reaction mixture was heated at 50° C. for 2h. The solvent was removed under vacuum and the residue was partitionedbetween ethyl acetate (120 mL) and water (100 mL). The organic phase waswashed with water (2×100 mL) and evaporated. The residue waschromatographed using a hexanes-ethyl acetate gradient, eluting theproduct at 6:4 hexanes-ethyl acetate, 4.83 g of 3-hydroxy-phthalic aciddimethyl ester, in 76% yield; ¹H NMR (CDCl₃) δ 3.89 (s, 3H), 3.92 (s,3H), 6.97 (dd, J=7.9 Hz, J=0.9 Hz, 1H), 7.09 (dd, J=8.6 Hz, J=1.0 Hz,1H), 7.46 (t, J=8.3 Hz, 1H), 10.58 (s, 1H).

Step 2:

Potassium carbonate (1.78 g, 12.9 mmol) and benzyl bromide (0.92 mL, 7.7mmol) were added to a stirred solution of 3-hydroxy-phthalic aciddimethyl ester (1.35 g, 6.40 mmol) in DMF (15 mL). The reaction mixturewas stirred overnight at room temperature and then quenched with coldwater (60 mL). The aqueous layer was extracted with ethyl acetate (3×40mL). The combined organic layers were washed with water (4×50 mL) andbrine (50 mL), dried (MgSO₄) and the solvent was evaporated undervacuum. The residue was chromatographed using a hexanes-ethyl acetategradient, eluting the product at 7:3 hexanes-ethyl acetate, 1.66 g of3-benzyloxy-phthalic acid dimethyl ester, in 86% yield; ¹H NMR (CDCl₃) δ3.89 (s, 3H), 3.95 (s, 3H), 5.16 (s, 2H), 7.15 (d, J=8.4 Hz, 1H),7.28-7.41 (m, 6H), 7.62 (d, J=7.9 Hz, 1H).

Step 3:

A mixture of 3-benzyloxy-phthalic acid dimethyl ester (1.64 g, 5.50mmol) and 3N NaOH (50 mL) in ethanol (100 mL) was heated to reflux for 1h and cooled to room temperature. The solvent was removed under vacuumand the residue was dissolved in water (100 mL), washed with CH₂Cl₂(2×100 mL) and acidified with 6N HCl to pH 1-2. The precipitate wasfiltered and washed with water (100 mL) to give 3-benzyloxy-phthalicacid as a white solid (1.10 g, 74% yield); ¹H NMR (DMSO-d₆) δ 5.20 (s,2H), 7.29-7.51 (m, 8H), 13.02 (br, 2H).

Step 4:

A mixture of 3-benzyloxy-phthalic acid (1.08 g, 4.00 mmol) andrac-α-aminoglutarimide hydrochloride (0.65 g, 4.0 mmol) in pyridine (10mL) was heated to reflux for 4 h. The reaction mixture was cooled andthe solvent was evaporated under vacuum. The residue was suspended inethyl acetate (200 mL) and washed with dilute aqueous HCl (100 mL). Theorganic phase was combined with the insoluble precipitate and evaporatedto dryness. The resulting solid was triturated with ethyl acetate (100mL), filtered, washed with additional ethyl acetate (50 mL), and driedto give the title compound (1.66 g, 86% yield); mp 238-240° C.; HPLC,Waters Symmetry C-18, 3.9×150 mm, 5 μm, 1 mL/min, 240 nm, 40/60CH₃CN/0.1% H₃PO₄, 7.23 (96.71%); ¹H NMR (DMSO-d₆) δ 1.99-2.08 (m, 1H),2.45-2.62 (m, 2H), 2.81-2.94 (m, 1H), 5.10 (dd, J=12.6 Hz, J=5.3 Hz,1H), 5.38 (s, 2H), 7.32-7.53 (m, 6H), 7.60 (d, J=8.5 Hz, 1H), 7.83 (t,J=8.2 Hz, 1H), 11.12 (s, 1H); ¹³C NMR (DMSO-d₆) δ 22.0, 30.9, 48.8,70.0, 115.6, 116.6, 120.2, 127.3, 128.0, 128.5, 133.3, 136.2, 137.0,155.5, 165.3, 166.8, 169.9, 172.8; Anal. Calcd for C₂₀H₁₆N₂O₅: C, 65.93;H, 4.43; N, 7.69. Found: C, 65.54; H, 4.35; N, 7.63.

Example 44-(3-Chloro-benzyloxy)-2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dione

Step 1:

To a stirred suspension of 3-hydroxyphthalic acid dimethyl ester (1.3 g,6.3 mmol) in acetone (20 mL) and potassium carbonate (2.1 g, 15.2 mmol)was added 3-chlorobenzyl bromide (1.0 mL, 7.6 mmol) and refluxedovernight. The solvent was evaporated and the residue was partitionedbetween water (100 mL) and ethyl acetate (150 mL), and washed with water(2×100 mL). The combined organic phases was dried, concentrated andpurified by flash column chromatography (EtOAc/Hexane) to give3-(3-chloro-benzyloxy)-phthalic acid dimethyl ester (1.9 g, 92% yield).The product was used in the next step without further purification.

Step 2:

A solution of 3-(3-chloro-benzyloxy)-phthalic acid dimethyl ester (1.9g, 5.8 mmol) in reagent alcohol (100 mL) and 3 N sodium hydroxide (60mL) was refluxed for two hours. The solution was evaporated and theresidue was dissolved in water (100 mL) and washed with methylenechloride (3×100 mL) then acidified to pH around 4. The resulting mixturewas extracted with ethyl acetate (2×100 mL) and the combined organiclayers was washed with water (2×100 mL), dried and concentrated to give3-(3-chloro-benzyloxy)-phthalic acid as an off-white solid (1.6 g, 87%yield). The product was used in the next step without furtherpurification.

Step 3:

A mixture of 3-(3-chloro-benzyloxy)-phthalic acid (1.6 g, 5.1 mmol),alpha-amino-glutarimide hydrochloride (0.87 g, 5.3 mmol) in pyridine wasrefluxed overnight. The mixture was evaporated and the residue waspurified by flash column chromatography (methanol/methylene chloride) togive4-(3-chloro-benzyloxy)-2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dioneas a white solid (0.74 g, 37% yield); HPLC: Waters Symmetry C₁₈, 3.9×150mm, 1 mL/min, 240 nm, 50/50 CH₃CN/0.1% H₃PO₄, 6.44 min (99.8%); mp,249-251° C.; ¹H NMR (DMSO-d₆) □ δ 2.02-2.07 (m, 1H, CHH), 2.54-2.62 (m,2H, CH₂), 2.83-2.91 (m, 1H, CHH), 5.12 (dd, J=6, 12 Hz, 1H, CH), 5.39(s, 2H, CH₂), 7.40-7.87 (m, 7H, Ar), 11.12 (s, 1H, NH); ¹³C NMR(DMSO-d₆) δ 21.96, 30.92, 48.77, 69.05, 115.72, 116.69, 120.10, 125.65,126.85, 127.84, 130.42, 133.18, 133.26, 137.07, 138.76, 155.19, 165.32,166.74, 169.89, 172.75. Anal Calcd For C₂₀H₁₅N₂O₅Cl+0.1H₂O: C, 59.96; H,3.82; N, 6.99; Cl, 8.85. Found: C, 59.93; H, 3.54; N, 6.91; Cl, 9.00.

Example 54-(4-Chloro-benzyloxy)-2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dione

Step 1:

To a stirred suspension of 3-hydroxyphthalic acid dimethyl ester (1.3 g,6.3 mmol) in acetone (20 mL) and potassium carbonate (2.6 g, 19 mmol)was added 4-chlorobenzyl chloride (1.1 g, 6.6 mmol) and refluxedovernight. The solvent was evaporated and the residue was partitionedbetween water (100 mL) and ethyl acetate (150 mL) and washed with water(2×100 mL). The combined organic phases was dried, concentrated andpurified by flash column chromatography (EtOAc/Hexane) to give3-(4-chloro-benzyloxy)-phthalic acid dimethyl ester (2.3 g, 110% crudeyield). The product was used in the next step without furtherpurification.

Step 2:

A solution of 3-(4-chloro-benzyloxy)-phthalic acid dimethyl ester (2.2g, 6.3 mmol) in reagent alcohol (100 mL) and 3 N sodium hydroxide (60mL) was refluxed for two hours. The solution was evaporated and theresidue was dissolved in water (100 mL) and washed with methylenechloride (3×100 mL) then acidified to pH around 4. The resulting mixturewas extracted with ethyl acetate (2×100 mL) and the combined organiclayers was washed with water (2×100 mL), dried and concentrated to give3-(4-chloro-benzyloxy)-phthalic acid as an off-white solid (1.9 g, 98%yield). The product was used in the next step without furtherpurification.

Step 3:

A mixture of 3-(4-chloro-benzyloxy)-phthalic acid (1.9 g, 6.2 mmol),alpha-amino-glutarimide hydrochloride (1.1 g, 6.5 mmol) in pyridine wasrefluxed overnight. The mixture was evaporated and the residue waspurified by flash column chromatography (methanol/methylene chloride) togive4-(4-chloro-benzyloxy)-2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dioneas a white solid (1.2 g, 49% yield); HPLC: Waters Symmetry C₁₈, 5 μm,3.9×150 mm, 1 mL/min, 240 nm, 50/50 CH₃CN/0.1% H₃PO₄, 6.64 min (99.9%);mp, 239-241° C.; ¹H NMR (DMSO-d₆) δ 2.00-2.07 (m, 1H, CHH), 2.54-2.62(m, 2H, CH₂), 2.83-2.95 (m, 1H, CHH), 5.12 (dd, J=6, 12 Hz, 1H, CH),5.38 (s, 2H, CH₂), 7.48-7.86 (m, 7H, Ar), 11.12 (s, 1H, NH); ¹³C NMR(DMSO-d₆) δ 21.96, 30.91, 48.75, 69.18, 115.66, 116.65, 120.16, 128.50,129.02, 132.52, 133.25, 135.22, 137.03, 155.27, 165.30, 166.74, 169.89,172.75. Anal Calcd For C₂₀H₁₅N₂O₅Cl: C, 60.24; H, 3.79; N, 7.02; Cl,8.89. Found: C, 60.41; H, 3.63; N, 7.02; Cl, 8.72.

Example 64-(3,4-Dichloro-benzyloxy)-2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dione

Step 1:

Triethylamine (2.70 mL, 19.4 mmol) was added to a mixture of3-hydroxyphthalic anhydride (3.00 g, 18.3 mmol) andrac-α-aminoglutarimide hydrochloride (3.01 g, 18.3 mmol) in DMF (60 mL).The reaction mixture was heated to 90° C. overnight, then cooled to roomtemperature and the solvent was evaporated under vacuum. The residue wasstirred in CH₂Cl₂ (100 mL) for 30 min and the solvent was removed undervacuum. The residue was stirred in water (120 mL) for 2 h and theresulting solid was filtered, washed with water (50 mL) and dried.1,4-Dioxane (200 mL) was added, and the resulting suspension was stirredfor 16 h and filtered; the insoluble material was reserved. The filtratewas treated with decolorizing carbon (2 g) and heated to reflux for 1 h.After cooling to 50° C. the reaction mixture was filtered through Celiteand the filter was washed with additional 1,4-dioxane (50 mL). Thefiltrate was combined with the insoluble precipitate and evaporated todryness. The resulting solid was triturated with ethyl acetate (100 mL),filtered and dried to give2-(2,6-dioxo-piperidin-3-yl)-4-hydroxy-isoindole-1,3-dione, 4.18 g, in56% yield; ¹H NMR (DMSO-d₆) δ 1.99-2.06 (m, 1H), 2.45-2.61 (m, 2H),2.82-2.96 (m, 1H), 5.08 (dd, J=12.6 Hz, J=5.3 Hz, 1H), 7.23-7.33 (m,2H), 7.66 (dd, J=8.2 Hz, J=7.2 Hz, 1H), 11.10 (s, 1H), 11.19 (s, 1H).

Step 2:

A mixture of polymer-supported triphenylphosphine (1.46 g, ˜4.4 mmol)and 3,4-dichlorobenzyl alcohol (0.65 g, 3.6 mmol) was stirred in THF (10mL) at 0° C. Keeping the reaction mixture at 0° C., a solution ofdiisopropylazodicarboxylate (0.87 mL, 4.4 mmol) in THF (2.1 mL) wasadded dropwise.2-(2,6-Dioxo-piperidin-3-yl)-4-hydroxy-isoindole-1,3-dione (1.00 g, 3.60mmol) was then added as a solid, the reaction mixture was stirred at 0°C. for 1 h and then at room temperature overnight. The solvent wasevaporated under vacuum and the residue was chromatographed using amethanol-CH₂Cl₂ gradient, eluting the product at 95:5 CH₂Cl₂-methanol.This material was dissolved in ethyl acetate (150 mL) and water (100 mL)was added. The organic phase was then washed with 10% dilute aqueoussodium carbonate (2×50 mL) and water (3×50 mL). The solvent was removedunder vacuum and the resulting solid was triturated with ether, filteredand dried to provide the title compound as a white solid (0.41 g, 26%yield); mp 245-247° C.; HPLC, Waters Symmetry C-18, 3.9×150 mm, 5 μm, 1mL/min, 240 nm, 60/40 CH₃CN/0.1% H₃PO₄, 3.50 (99.78%); ¹H NMR (DMSO-d₆)δ 2.02-2.07 (m, 1H), 2.45-2.62 (m, 2H), 2.82-2.96 (m, 1H), 5.12 (dd,J=12.6 Hz, J=5.4 Hz, 1H), 5.38 (s, 2H), 7.49-7.51 (m, 2H), 7.57 (d,J=8.5 Hz, 1H), 7.71 (d, J=8.2 Hz, 1H), 7.81-7.88 (m, 2H), 11.12 (s, 1H);¹³C NMR (DMSO-d₆) δ 22.0, 30.9, 48.8, 68.5, 115.9, 116.8, 120.1, 127.3,129.0, 130.5, 130.8, 131.2, 133.3, 137.1, 137.5, 155.1, 165.3, 166.8,169.9, 172.8; Anal. Calcd for C₂₀H₁₄N₂O₅Cl₂+0.3H₂O: C, 54.76; H, 3.35;N, 6.39. Found: C, 54.48; H, 3.07; N, 6.29.

Example 74-(3,5-Dichloro-benzyloxy)-2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dione

Step 1:

To a stirred suspension of 3-hydroxyphthalic acid dimethyl ester (0.5 g,2.4 mmol), (3,5-dichloro-phenyl)-methanol (0.84 g, 4.8 mmol), andpolymer-supported triphenyl phosphine (1.5 g, 4.8 mmol) in THF (30 mL)in ice-bath was slowly added diisopropyl azodicarboxylate (1.0 mL, 4.8mmol) and stirred at r.t. overnight. The mixture was filtered and thesolid was washed with ethyl acetate (10 mL). The filtrate was evaporatedand the residue was purified by flash column chromatography(EtOAc/Hexane) to give 3-(3,5-dichloro-benzyloxy)-phthalic acid dimethylester (0.42 g, 48% yield). The product was used in the next step withoutfurther purification.

Step 2:

A solution of 3-(3,5-dichloro-benzyloxy)-phthalic acid dimethyl ester(0.42 g, 1.2 mmol) in reagent alcohol (10 mL) and 3 N sodium hydroxide(10 mL) was refluxed for two hours. The solution was evaporated and theresidue was dissolved in water (10 mL) and washed with methylenechloride (2×10 mL) then acidified to pH around 4. The resulting mixturewas extracted with ethyl acetate (2×10 mL) and the combined organiclayers was washed with water (2×10 mL), dried and concentrated to give3-(3,5-dichloro-benzyloxy)-phthalic acid as an off-white solid (0.34 g,88% yield). The product was used in the next step without furtherpurification.

Step 3:

A mixture of 3-(3,5-dichloro-benzyloxy)-phthalic acid (0.3 g, 0.9 mmol),alpha-amino-glutarimide hydrochloride (0.15 g, 0.92 mmol) in pyridine(10 mL) was refluxed overnight. The mixture was evaporated and theresidue was purified by flash column chromatography (methanol/methylenechloride) to give4-(3,5-dichloro-benzyloxy)-2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dioneas a white solid (0.30 g, 84% yield); HPLC: Waters Symmetry C₁₈, 5 μm,3.9×150 mm, 1 mL/min, 240 nm, 70/30 CH₃CN/0.1% H₃PO₄, 3.6 min (98.0%);mp, 278-280° C.; ¹HNMR (DMSO-d₆) δ 2.01-2.08 (m, 1H, CHH), 2.54-2.63 (m,2H, CH₂), 2.84-2.96 (m, 1H, CHH), 5.13 (dd, J=6, 12 Hz, 1H, CH), 5.40(s, 2H, CH₂), 7.50-7.89 (m, 6H, Ar), 11.12 (s, 1H, NH); ¹³C NMR(DMSO-d₆) δ 21.95, 30.92, 48.79, 68.37, 115.90, 116.79, 120.02, 125.58,127.45, 133.25, 134.18, 137.14, 140.61, 154.93, 165.34, 166.73, 169.88,172.73. Anal Calcd For C₂₀H₁₄N₂O₅Cl₂: C, 55.45; H, 3.26; N, 6.47; Cl,16.37. Found: C, 55.20; H, 3.13; N, 6.38; Cl, 16.63.

Example 84-(3-Fluoro-benzyloxy)-2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dione

Step 1:

To a stirred suspension of 3-hydroxyphthalic acid dimethyl ester (1.4 g,6.7 mmol) in acetone (30 mL) and potassium carbonate (2.8 g, 20 mmol)was added 3-fluorobenzyl bromide (0.89 mL, 7.0 mmol) and refluxedovernight. The solvent was evaporated and the residue was partitionedbetween water (100 mL) and ethyl acetate (150 mL) and washed with water(2×100 mL). The combined organic phases was dried, concentrated andpurified by flash column chromatography (EtOAc/Hexane) to give3-(3-fluoro-benzyloxy)-phthalic acid dimethyl ester (2.4 g, 113% crudeyield). The product was used in the next step without furtherpurification.

Step 2:

A solution of 3-(3-fluoro-benzyloxy)-phthalic acid dimethyl ester (2.4 gcrude, 6.7 mmol) in reagent alcohol (100 mL) and 3 N sodium hydroxide(60 mL) was refluxed for two hours. The solution was evaporated and theresidue was dissolved in water (100 mL) and washed with methylenechloride (3×100 mL) then acidified to pH around 4. The resulting mixturewas extracted with ethyl acetate (2×100 mL) and the combined organiclayers was washed with water (2×100 mL), dried and concentrated to give3-(3-fluoro-benzyloxy)-phthalic acid as an off-white solid (1.9 g, 101%crude yield). The product was used in the next step without furtherpurification.

Step 3:

A mixture of 3-(3-fluoro-benzyloxy)-phthalic acid (1.9 g, 6.7 mmol),alpha-amino-glutarimide hydrochloride (1.2 g, 7.0 mmol) in pyridine wasrefluxed overnight. The mixture was evaporated and the residue waspurified by flash column chromatography (methanol/methylene chloride) togive4-(3-fluoro-benzyloxy)-2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dioneas a white solid (2.3 g, 89% yield); HPLC: Waters Symmetry C₁₈, 5 μm,3.9×150 mm, 1 mL/min, 240 nm, 60/40 CH₃CN/0.1% H₃PO₄, 2.22 min (99.9%);mp, 241-243° C.; ¹H NMR (DMSO-d₆) δ 2.01-2.08 (m, 1H, CHH), 2.55-2.62(m, 2H, CH₂), 2.83-2.95 (m, 1H, CHH), 5.11 (dd, J=6, 12 Hz, 1H, CH),5.40 (s, 2H, CH₂), 7.15-7.87 (m, 7H, Ar), 11.12 (s, 1H, NH); ¹³C NMR(DMSO-d₆) δ 21.96, 30.92, 48.77, 69.11, 113.61, 113.90, 114.52, 114.80,115.71, 116.70, 120.13, 122.96, 122.99, 130.49, 130.60, 133.25, 137.05,139.08, 139.18, 155.21, 160.59, 163.81, 165.33, 166.74, 169.89, 172.73.Anal Calcd For C₂₀H₁₅N₂O₅F: C, 62.83; H, 3.95; N, 7.33; F 4.97. Found:C, 62.72; H, 3.75; N, 7.27; F, 5.02.

Example 94-(3-Bromo-benzyloxy)-2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dione

Step 1:

To a stirred suspension of 3-hydroxyphthalic acid dimethyl ester (1.3 g,6.2 mmol) in acetone (30 mL) and potassium carbonate (2.5 g, 18.4 mmol)was added 3-bromobenzyl bromide (1.6 g, 6.4 mmol) and refluxedovernight. The solvent was evaporated and the residue was partitionedbetween water (100 mL) and ethyl acetate (150 mL) and washed with water(2×100 mL). The combined organic phases was dried, concentrated andpurified by flash column chromatography (EtOAc/Hexane) to give3-(3-bromo-benzyloxy)-phthalic acid dimethyl ester (2.5 g, 109% crudeyield). The product was used in the next step without furtherpurification.

Step 2:

A solution of 3-(3-bromo-benzyloxy)-phthalic acid dimethyl ester (1.9 g,5.8 mmol) in reagent alcohol (100 mL) and 3 N sodium hydroxide (60 mL)was refluxed for two hours. The solution was evaporated and the residuewas dissolved in water (100 mL) and washed with methylene chloride(3×100 mL) then acidified to pH around 4. The resulting mixture wasextracted with ethyl acetate (2×100 mL) and the combined extracts waswashed with water (2×100 mL), dried and concentrated to give3-(3-bromo-benzyloxy)-phthalic acid as an off-white solid (2.5 g, 109%crude yield). The product was used in the next step without furtherpurification.

Step 3:

A mixture of 3-(3-bromo-benzyloxy)-phthalic acid (2.4 g, 6.7 mmol),alpha-amino-glutarimide hydrochloride (1.2 g, 7.1 mmol) in pyridine wasrefluxed overnight. The mixture was evaporated and the residue waspurified by flash column chromatography (methanol/methylene chloride) togive4-(3-bromo-benzyloxy)-2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dioneas a white solid (1.8 g, 62% yield); HPLC: Waters Symmetry C₁₈, 5 μm,3.9×150 mm, 1 mL/min, 240 nm, 60/40 CH₃CN/0.1% H₃PO₄, 3.55 min (99.9%);mp, 246-248° C.; ¹H NMR (DMSO-d₆) δ 2.03-2.08 (m, 1H, CHH), 2.54-2.62(m, 2H, CH₂), 2.83-2.95 (m, 1H, CHH), 5.12 (dd, J=6, 12 Hz, 1H, CH),5.39 (s, 2H, CH₂), 7.37-7.88 (m, 7H, Ar), 11.12 (s, 1H, NH); ¹³C NMR(DMSO-d₆) δ 21.96, 30.92, 48.77, 69.00, 115.72, 116.69, 120.10, 121.76,126.05, 129.75, 130.69, 130.74, 133.26, 137.07, 138.99, 155.19, 165.31,166.74, 169.89, 172.74. Anal Calcd For C₂₀H₁₅N₂O₅Br: C, 54.19; H, 3.41;N, 6.32; Br 18.03. Found: C, 54.02; H, 3.22; N, 6.27; Br, 17.81.

Example 102-(2,6-Dioxo-piperidin-3-yl)-4-(3-methyl-benzyloxy)-isoindole-1,3-dione

Step 1:

To a stirred suspension of 3-hydroxyphthalic acid dimethyl ester (1.4 g,6.4 mmol) in acetone (30 mL) and potassium carbonate (2.7 g, 19.3 mmol)was added 1-bromomethyl-3-methyl-benzene (0.91 mL, 6.7 mmol) andrefluxed overnight. The solvent was evaporated and the residue waspartitioned between water (100 mL) and ethyl acetate (150 mL) and washedwith water (2×100 mL). The combined organic phases was dried,concentrated and purified by flash column chromatography (EtOAc/Hexane)to give 3-(3-methyl-benzyloxy)-phthalic acid dimethyl ester (2.3 g, 115%crude yield). The product was used in the next step without furtherpurification.

Step 2:

A solution of 3-(3-methyl-benzyloxy)-phthalic acid dimethyl ester (2.0 gcrude, 6.4 mmol) in reagent alcohol (100 mL) and 3 N sodium hydroxide(60 mL) was refluxed for two hours. The solution was evaporated and theresidue was dissolved in water (100 mL) and washed with methylenechloride (3×100 mL) then acidified to pH around 4. The resulting mixturewas extracted with ethyl acetate (2×100 mL) and the combined organiclayers was washed with water (2×100 mL), dried and concentrated to give3-(3-methyl-benzyloxy)-phthalic acid as an off-white solid (3.0 g, 130%crude yield). The product was used in the next step without furtherpurification.

Step 3:

A mixture of 3-(3-methyl-benzyloxy)-phthalic acid (1.8 g, 6.4 mmol),alpha-amino-glutarimide hydrochloride (1.1 g, 6.8 mmol) in pyridine wasrefluxed overnight. The mixture was evaporated and the residue waspurified by flash column chromatography (methanol/methylene chloride) togive2-(2,6-dioxo-piperidin-3-yl)-4-(3-methyl-benzyloxy)-isoindole-1,3-dioneas a white solid (1.2 g, 48% yield); HPLC: Waters Symmetry C₁₈, 5 μm,3.9×150 mm, 1 mL/min, 240 nm, 60/40 CH₃CN/0.1% H₃PO₄, 3.16 min (99.9%);mp, 195-197° C.; ¹H NMR (DMSO-d₆) δ 2.00-2.07 (m, 1H, CHH), 2.33 (s, 3H,CH₃), 2.54-2.62 (m, 2H, CH₂), 2.83-2.95 (m, 1H, CHH), 5.10 (dd, J=6, 12Hz, 1H, CH), 5.33 (s, 2H, CH₂), 7.15-7.85 (m, 7H, Ar), 11.12 (s, 1H,NH); ¹³C NMR (DMSO-d₆) δ 21.00, 21.96, 30.92, 48.74, 70.08, 115.49,116.58, 120.19, 124.40, 127.85, 128.40, 128.60, 133.26, 136.03, 136.98,137.64, 155.53, 165.30, 166.77, 169.91, 172.75. Anal Calcd ForC₂₁H₁₈N₂O₅: C, 66.66; H, 4.79; N, 7.40. Found: C, 66.50; H, 4.79; N,7.34.

Example 112-(2,6-Dioxo-piperidin-3-yl)-4-(4-methanesulfonyl-benzyloxy)-isoindole-1,3-dione

Step 1:

To a stirred suspension of 3-hydroxyphthalic acid dimethyl ester (1.3 g,6.1 mmol) in acetone (25 mL) and potassium carbonate (2.5 g, 18 mmol)was added 1-bromomethyl-4-methanesulfonyl-benzene (1.6 g, 6.4 mmol) andrefluxed overnight. The solvent was evaporated and the residue waspartitioned between water (100 mL) and ethyl acetate (150 mL) and washedwith water (2×100 mL). The combined organic phases was dried,concentrated and purified by flash column chromatography (EtOAc/Hexane)to give 3-(4-methanesulfonyl-benzyloxy)-phthalic acid dimethyl ester(2.4 g, 104% crude yield). The product was used in the next step withoutfurther purification.

Step 2:

A solution of 3-(4-methanesulfonyl-benzyloxy)-phthalic acid dimethylester (2.3 g, 6.1 mmol) in reagent alcohol (140 mL) and 3 N sodiumhydroxide (70 mL) was refluxed for two hours. The solution wasevaporated and the residue was dissolved in water (100 mL) and washedwith methylene chloride (3×100 mL) then acidified to pH around 4. Theresulting mixture was extracted with ethyl acetate (2×100 mL) and thecombined organic layers was washed with water (2×100 mL), dried andconcentrated to give 3-(4-methanesulfonyl-benzyloxy)-phthalic acid as anoff-white solid (2.15 g, 101% crude yield). The product was used in thenext step without further purification.

Step 3:

A mixture of 3-(4-methanesulfonyl-benzyloxy)-phthalic (2.1 g, 6.1 mmol),alpha-amino-glutarimide hydrochloride (1.1 g, 6.4 mmol) in pyridine wasrefluxed overnight. The mixture was evaporated and the residue waspurified by flash column chromatography (methanol/methylene chloride) togive2-(2,6-dioxo-piperidin-3-yl)-4-(4-methanesulfonyl-benzyloxy)-isoindole-1,3-dioneas a white solid (1.3 g, 47% yield); HPLC: Waters Symmetry C₁₈, 5 μm,3.9×150 mm, 1 mL/min, 240 nm, 35/65 CH₃CN/0.1% H₃PO₄, 2.09 min (99.9%);mp, 293-295° C.; ¹H NMR (DMSO-d₆) δ 2.03-2.07 (m, 1H, CHH), 2.54-2.63(m, 2H, CH₂), 2.85-2.90 (m, 1H, CHH), 3.23 (s, 3H, CH₃SO₂), 5.11 (dd,J=6, 12 Hz, 1H, CH), 5.52 (s, 2H, CH₂), 7.49-8.00 (m, 7H, Ar), 11.13 (s,1H, NH); ¹³C NMR (DMSO-d₆) δ 21.97, 30.91, 43.46, 48.78, 69.09, 115.82,116.73, 120.11, 127.21, 127.62, 133.28, 137.10, 140.26, 142.17, 155.12,165.30, 166.73, 169.89, 172.75. Anal Calcd For C₂₁H₁₈N₂O₇S+0.2H₂O: C,56.55; H, 4.16; N, 6.28; S, 7.19. Found: C, 56.32; H, 3.80; N, 6.16; S,7.20.

Example 122-(2,6-Dioxo-piperidin-3-yl)-4-(3-methoxy-benzyloxy)-isoindole-1,3-dione

Step 1:

To a stirred suspension of 3-hydroxyphthalic acid dimethyl ester (1 g,5.2 mmol) in acetone (45 mL) and potassium carbonate (2.2 g, 15.7 mmol)was added 1-bromomethyl-3-methoxy-benzene (0.77 mL, 5.5 mmol) andrefluxed for three hours. The solvent was evaporated and the residue waspartitioned between water (50 mL) and ethyl acetate (80 mL) and washedwith water (2×50 mL). The combined organic phases was dried,concentrated and purified by flash column chromatography (EtOAc/Hexane)to give 3-(3-methoxy-benzyloxy)-phthalic acid dimethyl ester (2.1 g,118% crude yield). The product was used in the next step without furtherpurification.

Step 2:

A solution of 3-(3-methoxy-benzyloxy)-phthalic acid dimethyl ester (2.0g crude, 5.5 mmol) in reagent alcohol (100 mL) and 3 N sodium hydroxide(35 mL) was refluxed for two hours. The solution was evaporated and theresidue was dissolved in water (80 mL) and washed with methylenechloride (3×70 mL) then acidified to pH around 4. The resulting mixturewas extracted with ethyl acetate (2×60 mL) and the combined organiclayers was washed with water (2×70 mL), dried and concentrated to give3-(3-methoxy-benzyloxy)-phthalic acid as an off-white solid (1.6 g, 98%crude yield). The product was used in the next step without furtherpurification.

Step 3:

A mixture of 3-(3-methoxy-benzyloxy)-phthalic acid (1.5 g, 5.2 mmol),alpha-amino-glutarimide hydrochloride (0.89 g, 5.4 mmol) in pyridine wasrefluxed overnight. The mixture was evaporated and the residue waspurified by flash column chromatography (methanol/methylene chloride) togive2-(2,6-dioxo-piperidin-3-yl)-4-(3-methoxy-benzyloxy)-isoindole-1,3-dioneas a white solid (0.25 g, 12% yield); HPLC: Waters Symmetry C₁₈, 5 μm,3.9×150 mm, 1 mL/min, 240 nm, 60/40 CH₃CN/0.1% H₃PO₄, 2.41 min (99.1%);mp, 197-201° C.; ¹H NMR (DMSO-d₆) δ 2.02-2.06 (m, 1H, CHH), 2.59-2.62(m, 2H, CH₂), 2.83-2.90 (m, 1H, CHH), 3.77 (s, 3H, CH₃), 5.10 (dd, J=6,12 Hz, 1H, CH), 5.35 (s, 2H, CH₂), 6.89-7.85 (m, 7H, Ar), 11.11 (s, 1H,NH); ¹³C NMR (DMSO-d₆) δ 21.95, 30.92, 48.75, 55.01, 69.80, 112.72,113.27, 115.55, 116.66, 119.17, 120.21, 129.61, 133.25, 136.98, 137.74,155.42, 159.35, 165.32, 166.77, 169.90, 172.74. Anal Calcd ForC₂₁H₁₈N₂O₆+0.1; H₂O: C, 63.67; H, 4.63; N, 7.07. Found: C, 63.49; H,4.40; N, 7.00.

Example 134-(Benzo[1,3]dioxol-5-ylmethoxy)-2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dione

Step 1:

To a stirred suspension of 3-hydroxyphthalic acid dimethyl ester (1.0 g,4.8 mmol), benzo[1,3]dioxol-5-yl-methanol (1.4 g, 9.5 mmol), andpolymer-supported triphenyl phosphine (3.0 g, 9.5 mmol) in THF (30 mL)in an ice-bath was slowly added diisopropyl azodicarboxylate (1.9 mL,9.5 mmol) and stirred at r.t. overnight. The mixture was filtered andthe filter was washed with ethyl acetate (10 mL). The filtrate wasevaporated and the residue was purified by flash column chromatography(EtOAc/Hexane) to give 3-(benzo[1,3]dioxol-5-ylmethoxy)-phthalic aciddimethyl ester (1.7 g, 102% crude yield). The product was used in thenext step without further purification.

Step 2:

A solution of 3-(benzo[1,3]dioxol-5-ylmethoxy)-phthalic acid dimethylester (1.6 g crude, 4.8 mmol) in reagent alcohol (100 mL) and 3 N sodiumhydroxide (35 mL) was refluxed for two hours. The solution wasevaporated and the residue was dissolved in water (80 mL) and washedwith methylene chloride (3×70 mL) then acidified to pH around 4. Theresulting mixture was extracted with ethyl acetate (2×60 mL) and thecombined organic layers was washed with water (2×70 mL), dried andconcentrated to give 3-(benzo[1,3]dioxol-5-ylmethoxy)-phthalic acid asan off-white solid (1.2 g. 80% crude yield). The product was used in thenext step without further purification.

Step 3:

A mixture of 3-(benzo[1,3]dioxol-5-ylmethoxy)-phthalic acid (1.2 g, 3.8mmol), alpha-amino-glutarimide hydrochloride (0.66 g, 4.0 mmol) inpyridine was refluxed overnight. The mixture was evaporated and theresidue was purified by flash column chromatography (methanol/methylenechloride) to give4-(benzo[1,3]dioxol-5-ylmethoxy)-2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dioneas a white solid (0.45 g, 29% yield); HPLC: Waters Symmetry C₁₈, 5 μm.3.9×150 mm, 1 mL/min, 240 nm, 50/50 CH₃CN/0.1% H₃PO₄, 4.06 min (98.6%);mp, 229-231° C.; ¹H NMR (DMSO-d₆) δ 2.02-2.05 (m, 1H, CHH), 2.55-2.62(m, 2H, CH₂), 2.82-2.94 (m, 1H, CHH), 5.10 (dd, J=6, 12 Hz, 1H, CH),5.26 (s, 2H, CH₂), 6.03 (s, 2H, CH₂), 6.93-7.85 (m, 6H, Ar), 11.11 (s,1H, NH); ¹³C NMR (DMSO-d₆) δ 21.95, 30.91, 48.74, 70.00, 101.05, 108.10,108.18, 115.49, 116.61, 120.28, 121.25, 129.78, 133.24, 136.95, 147.04,147.38, 155.44, 165.30, 166.76, 169.89, 172.74. Anal Calcd ForC₂₁H₁₆N₂O₇: C, 61.77; H, 3.95; N, 6.86. Found: C, 61.44; H, 3.72; N,6.79.

Example 142-(2,6-Dioxo-piperidin-3-yl)-4-(naphthalene-2-ylmethoxy)-isoindole-1,3-dione

A mixture of triphenylphosphine (1.15 g, 4.40 mmol) and2-naphthalenemethanol (0.58 g, 3.6 mmol) was stirred in THF (10 mL) at0° C. Keeping the reaction mixture at 0° C., a solution ofdiisopropylazodicarboxylate (0.87 mL, 4.4 mmol) in THF (2.1 mL) wasadded dropwise.2-(2,6-Dioxo-piperidin-3-yl)-4-hydroxy-isoindole-1,3-dione (1.00 g, 3.60mmol) was then added as a solid, the reaction mixture was stirred at 0°C. for 1 h and then at room temperature overnight. The precipitate wasfiltered, washed with additional THF (10 mL) and dried. The resultingsolid was stirred in hexane (50 mL) for 2 h, filtered and dried. Theresulting solid was heated to reflux in methanol (50 mL) for 1 h,filtered and dried to afford the product as a white solid (0.46 g, 30%yield); mp>260° C.; HPLC, Waters Symmetry C-18, 3.9×150 mm, 5 μm, 1mL/min, 240 nm, 50/50 CH₃CN/0.1% H₃PO₄, 6.20 (99.48%); ¹H NMR (DMSO-d₆)δ 2.02-2.07 (m, 1H), 2.44-2.62 (m, 2H), 2.82-2.97 (m, 1H), 5.12 (dd,J=12.5 Hz, J=5.3 Hz, 1H), 5.54 (s, 2H), 7.46-8.05 (m, 10H), 11.13 (s,1H); ¹³C NMR (DMSO-d₆) δ 22.0, 31.0, 48.8, 70.3, 115.6, 116.7, 120.4,125.3, 126.1, 126.3, 126.4, 127.7, 127.8, 128.2, 132.6, 132.7, 133.3,133.8, 137.0, 155.6, 165.4, 166.8, 169.9, 172.8; Anal. Calcd forC₂₄H₁₈N₂O₅: C, 69.39; H, 4.02; N, 6.61. Found: C, 69.56; H, 4.38; N,6.76.

Example 152-(2,6-Dioxo-piperidin-3-yl)-4-(quinolin-3-ylmethoxy)-isoindole-1,3-dione

Step 1:

3-Quinolinecarbaldehyde (2.00 g, 12.7 mmol) was dissolved in 25 mL ofmethanol. To this solution was added sodium borohydride (0.24 g, 6.4mmol) in small portions over a period of 20 minutes. Then 2 mL of waterwere added and the mixture was evaporated. The residue was dissolved inethyl acetate (75 mL) and washed with water (3×75 mL), dried (MgSO₄) andevaporated, providing 1.8 g of quinolin-3-yl-methanol in 90% yield; ¹HNMR (DMSO-d₆) δ 4.89 (s, 2H), 7.53 (t, J=7.1 Hz, 1H), 7.64-7.71 (m, 1H),7.77 (d, J=8.2 Hz, 1H), 8.04-8.12 (m, 2H), 8.83 (d, J=2.0 Hz, 1H).

Step 2:

A mixture of polymer-supported PPh₃ (3.1 g, ˜9.5 mmol) andquinolin-3-yl-methanol (0.76 g, 4.8 mmol) in 20 mL THF was cooled to 0°C. under N₂. Diisopropyl azodicarboxylate (1.9 g, 9.5 mmol) was addeddropwise, and subsequently 3-hydroxyphthalic acid dimethyl ester (1.0 g,4.8 mmol) was added as a solid. The mixture stirred for an additionalhour at 0° C. and was then allowed to warm to room temperature. Afterstirring for 16 h, the mixture was filtered. The filter was washed withethyl acetate (25 mL) and the combined filtrates were evaporated.

Step 3:

The crude product from Step 2 was dissolved in a mixture of 3N NaOH (50mL) and ethanol (100 mL), and the resulting solution was heated toreflux for 2 h. The mixture was cooled and the solvent was removed undervacuum. The residue was dissolved in water (100 mL) and washed withCH₂Cl₂ (3×100 mL), acidified to pH 2-3 (HCl). The resulting precipitatewas filtered and washed with additional water and then ethyl acetate,and dried under vacuum.

Step 4:

The crude product from Step 3 and rac-α-aminoglutarimide hydrochloride(0.78 g, 4.8 mmol) in pyridine (10 mL) was heated to reflux for 16 h.The mixture was cooled and evaporated under vacuum. The residue waschromatographed using a CH₂Cl₂-methanol gradient, eluting the titlecompound at 95:5 CH₂Cl₂-methanol, 0.37 g, in 20% yield over 3 steps; mp263-265° C.; HPLC, Waters Symmetry C-18, 3.9×150 mm, 5 μm, 1 mL/min, 240nm, 40/60 CH₃CN/water, 3.75 (97.84%); ¹H NMR (DMSO-d₆) δ 2.02-2.06 (m,1H), 2.55-2.62 (m, 2H), 2.81-2.90 (m, 1H), 5.11 (dd, J=12.3 Hz, J=5.3Hz, 1H), 5.61 (s, 2H), 7.51 (d, J=7.2 Hz, 1H), 7.62-7.71 (m, 2H),7.77-7.90 (m, 2H), 8.00-8.08 (m, 2H), 8.48 (s, 1H), 9.04 (d, J=1.8 Hz,1H), 11.12 (s, 1H); ¹³C NMR (DMSO-d₆) δ 22.0, 30.9, 48.8, 68.3, 115.9,116.8, 120.4, 127.1, 127.2, 128.1, 128.8, 129.3, 129.8, 133.3, 134.5,137.1, 147.3, 150.3, 155.3, 165.3, 166.8, 169.9, 172.8; Anal. Calcd forC₂₃H₁₇N₃O₅.0.4H₂O: C, 65.37; H, 4.25; N, 9.94. Found: C, 65.35; H, 4.06;N, 9.92.

Example 162-(2,6-Dioxo-piperidin-3-yl)-4-(quinolin-2-ylmethoxy)-isoindole-1,3-dione

Step 1:

2-Quinolinecarbaldehyde (2.00 g, 12.7 mmol) was dissolved in 25 mL ofmethanol. To this solution was added sodium borohydride (0.24 g, 6.4mmol) in small portions over a period of 20 minutes. Then 2 mL of waterwere added and the mixture was evaporated. The residue was dissolved inethyl acetate (75 mL) and washed with water (3×75 mL) and evaporated.The residue was chromatographed in CH₂Cl₂-methanol gradient, eluting theproduct at 97:3 CH₂Cl₂-methanol, and providing 1.7 g ofquinolin-2-yl-methanol in 85% yield; ¹H NMR (CDCl₃) δ 4.92 (s, 2H), 7.26(d, J=2.1 Hz, 1H), 7.30-7.57 (m, 1H), 7.68-7.75 (m, 1H), 7.82 (dd, J=8.0Hz, J=0.9 Hz, 1H), 8.07 (d, J=8.4 Hz, 1H), 8.13 (d, J=8.5 Hz, 1H).

Step 2:

A mixture of polymer-supported PPh₃ (3.1 g, ˜9.5 mmol) andquinolin-2-yl-methanol (0.76 g, 4.8 mmol) in 20 mL THF was cooled to 0°C. under N₂. Diisopropylazodicarboxylate (1.9 g, 9.5 mmol) was addeddropwise, and subsequently 3-hydroxy-phthalic acid dimethyl ester (1.0g, 4.8 mmol) was added as a solid. The mixture stirred for an additionalhour at 0° C. and was then allowed to warm to room temperature. Afterstirring for 16 h, the mixture was filtered. The filter was washed withethyl acetate (25 mL) and the combined filtrates were evaporated.

Step 3:

The crude product from Step 2 was dissolved in a mixture of 3N NaOH (50L) and ethanol (100 mL), and the resulting solution was heated to refluxfor 2 h. The mixture was cooled and the solvent was removed undervacuum. The residue was dissolved in water (100 mL) and washed withCH₂Cl₂ (3×100 mL), acidified to pH 2-3 (HCl). The resulting precipitatewas filtered and washed with additional water and then ethyl acetate,and dried under vacuum.

Step 4:

The product from Step 3 and rac-α-aminoglutarimide hydrochloride (0.78g, 4.8 mmol) in pyridine (10 mL) was heated to reflux for 16 h. Themixture was cooled and evaporated under vacuum. The residue waschromatographed using a CH₂Cl₂-methanol gradient, eluting the product at95:5 CH₂Cl₂-methanol. This material was triturated in DMF (5 mL),filtered and washed with additional 2 mL of DMF, and dried under vacuum.This material was then purified by preparative HPLC using a mobile phaseof 35/65 acetonitrile-water, providing 75 mg of the title compound in 4%yield over 3 steps; mp 254-256° C.; HPLC, Waters Symmetry C-18, 3.9×150mm, 5 μm, 1 mL/min, 240 nm, 30/70 CH₃CN/0.1% H₃PO₄, 7.02 (94.00%); ¹HNMR (DMSO-d₆) δ 2.03-2.08 (m, 1H), 2.57-2.64 (m, 2H), 2.85-2.96 (m, 1H),5.13 (dd, J=12.2 Hz, J=4.9 Hz, 1H), 5.62 (s, 2H), 7.50 (d, J=7.1 Hz,1H), 7.62-7.66 (m, 2H), 7.77-7.87 (m, 3H), 8.00-8.03 (m, 2H), 8.48 (d,J=8.5 Hz, 1H), 11.13 (s, 1H); ¹³C NMR (DMSO-d₆) δ 22.0, 31.0, 48.8,71.4, 115.8, 116.8, 119.1, 120.3, 126.7, 127.2, 128.0, 128.5, 130.0,133.3, 137.1, 137.2, 146.9, 155.3, 156.7, 165.4, 166.8, 169.9, 172.8;Anal. Calcd for C₂₃H₁₇N₃O₅.1.6H₂O: C, 62.19; H, 4.58; N, 9.46. Found: C,62.20; H, 3.97; N, 9.15.

Example 174-(Benzofuran-2-ylmethoxy)-2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dione

Step 1:

2-Benzofurancarbaldehyde (2.2 g, 15 mmol) was dissolved in 25 mL ofmethanol. To this solution was added sodium borohydride (0.28 g, 7.5mmol) in small portions over a period of 20 minutes. Then 2 mL of waterwere added and the mixture was evaporated. The residue was dissolved inethyl acetate (75 mL) and washed with water (3×75 mL), dried (MgSO₄) andevaporated, providing 2.1 g of benzofuran-2-yl-methanol, in 95% yield;¹H NMR (CDCl₃) δ 2.03 (t, J=6.1 Hz, 1H), 4.77 (d, J=6.1 Hz, 214), 6.66(s, 1H), 7.19-7.32 (m, 2H), 7.45-7.50 (m, 1H), 7.53-7.57 (m, 1H).

Step 2:

A mixture of polymer-supported PPh₃ (3.1 g, ˜9.5 mmol) andbenzofuran-2-yl-methanol (0.70 g, 4.8 mmol) in 20 mL THF was cooled to0° C. under N₂. Diisopropylazodicarboxylate (1.9 g, 9.5 mmol) was addeddropwise, and subsequently 3-hydroxy-phthalic acid dimethyl ester (1.0g, 4.8 mmol) was added as a solid. The mixture stirred for an additionalhour at 0° C. and was then allowed to warm to room temperature. Afterstirring for 16 h, the mixture was filtered. The filter was washed withethyl acetate (25 mL) and the combined filtrates were evaporated. Theresidue was dissolved in 75 mL ethyl acetate and washed with Na₂CO₃(2×75 mL) and water (2×75 mL), dried (MgSO₄), and evaporated.

Step 3:

The crude product from Step 2 was dissolved in a mixture of 3N NaOH (50mL) and ethanol (100 mL), and the resulting solution was heated toreflux for 2 h. The mixture was cooled and the solvent was removed undervacuum. The residue was dissolved in water (100 mL) and washed withCH₂Cl₂ (3×100 mL), acidified (HCl), and extracted with ethyl acetate(3×75 mL). The combined organic extracts were washed with water (3×75mL), dried (MgSO₄) and evaporated, providing 0.86 g of3-(benzofuran-2-ylmethoxy)-phthalic acid, in 57% yield over two steps:¹H NMR (DMSO-d₆) δ 5.35 (s. 2H), 7.04 (s, 1H), 7.22-7.37 (m, 2H),7.45-7.67 (m, 5H).

Step 4:

A mixture of 3-(benzofuran-2-ylmethoxy)-phthalic acid (0.55 g, 1.8 mmol)and rac-α-aminoglutarimide hydrochloride (0.30 g, 1.8 mmol) in pyridine(10 mL) was heated to reflux for 16 h. The mixture was cooled andevaporated under vacuum. The residue was dissolved in CH₂Cl₂ (100 mL)and washed with dilute aqueous HCl (2×100 mL) and water (2×100 mL) andwas evaporated. The residue was chromatographed using a CH₂Cl₂-methanolgradient, eluting the title compound at 95:5 CH₂Cl₂-methanol, 0.46 g, in65% yield; mp 234-236° C.; HPLC, Waters Symmetry C-18, 3.9×150 mm, 5 μm,1 mL/min, 240 nm, 50/50 CH₃CN/0.1% H₃PO₄, 4.16 (98.58%); ¹H NMR(DMSO-d₆) δ 1.99-2.04 (m, 1H), 2.43-2.61 (m, 2H), 2.81-2.95 (m, 1H),5.08 (dd, J=12.7 Hz, J=5.3 Hz, 1H), 5.55 (s, 2H), 7.14 (s, 1H),7.23-7.37 (m, 2H), 7.50 (d, J=7.0 Hz, 1H), 7.60 (d, J=8.1 Hz, 1H), 7.68(d, J=7.5 Hz, 1H), 7.74 (d, J=8.5 Hz, 1H), 7.86 (t, J=7.8 Hz, 1H), 11.11(s, 1H); ¹³C NMR (DMSO-d₆) δ 23.9, 30.9, 48.8, 63.1, 107.4, 111.3,115.9, 116.7, 120.2, 121.6, 123.1, 125.0, 127.5, 133.3, 137.0, 152.0,154.6, 155.0, 165.2, 166.7, 169.9, 172.7; Anal. Calcd forC₂₂H₁₆N₂O₆.0.15H₂O: C, 64.91; H, 4.04; N, 6.88. Found: C, 64.89; H,3.99; N, 6.84.

Example 184-(Benzo[b]thiophen-2-ylmethoxy)-2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dione

Step 1:

A mixture of polymer-supported PPh₃ (3.1 g, ˜9.5 mmol) and1-benzothiophen-2-yl-methanol (1.0 g, 6.1 mmol) in 20 mL THF was cooledto 0° C. under N₂. Diisopropylazodicarboxylate (1.9 g, 9.5 mmol) wasadded dropwise, and subsequently 3-hydroxy-phthalic acid dimethyl ester(1.0 g, 4.8 mmol) was added as a solid. The mixture stirred for anadditional hour at 0° C. and was then allowed to warm to roomtemperature. After stirring for 16 h, the mixture was filtered. Thefilter was washed with ethyl acetate (25 mL) and the combined filtrateswere evaporated.

Step 2:

The crude product from Step 1 was dissolved in a mixture of 3N NaOH (50mL) and ethanol (100 mL), and the resulting solution was heated toreflux for 2 h. The mixture was cooled and the solvent was removed undervacuum. The residue was dissolved in water (100 mL) and washed withCH₂Cl₂ (3×100 mL), acidified (HCl), and extracted into ethyl acetate(3×75 mL). The combined organic extracts were washed with water (3×75mL), dried (MgSO₄), and evaporated.

Step 3:

The crude product from Step 2 and rac-α-aminoglutarimide hydrochloride(0.40 g, 2.5 mmol) in pyridine (10 mL) was heated to reflux for 16 h.The mixture was cooled and evaporated under vacuum. The residue waschromatographed using a CH₂Cl₂-methanol gradient, eluting the product at95:5 CH₂Cl₂-methanol, and providing 0.40 g in 30% yield over 3 steps; mp247-249° C.; HPLC, Waters Symmetry C-18, 3.9×150 mm, 5 μm, 1 mL/min, 240nm, 50/50 CH₃CN/0.1% H₃PO₄, 5.68 (100.00%); ¹H NMR (DMSO-d₆) δ 2.01-2.06(m, 1H), 2.44-2.61 (m, 2H), 2.82-2.96 (m, 1H), 5.10 (dd, J=12.6 Hz,J=5.3 Hz, 1H), 5.71 (s, 2H), 7.32-7.42 (m, 2H), 7.49 (d, J=7.1 Hz, 1H),7.58 (s, 1H), 7.68 (d, J=8.5 Hz, 1H), 7.80-7.87 (m, 2H), 7.96 (d, J=8.2Hz, 1H), 11.11 (s, 1H); ¹³C NMR (DMSO-d₆) δ 22.0, 30.9, 48.8, 66.1,115.9, 116.9, 120.4, 122.6, 123.8, 123.9, 124.5, 124.7, 133.3, 136.9,138.9, 139.5, 154.9, 165.2, 166.7, 169.9, 172.8; Anal. Calcd forC₂₂H₁₆N₂O₅S: C, 62.85; H, 3.84; N, 6.66. Found: C, 62.88; H, 3.46; N,6.57.

Example 192-(2,6-Dioxo-piperidin-3-yl)-4-(furan-2-ylmethoxy)-isoindole-1,3-dione

To a solution of triphenylphosphine (630 mg, 2.4 mmol) andfuran-2-yl-methanol (0.17 mL, 2.0 mmol) in THF (10 mL) was added asolution of DEAD (0.38 mL, 2.4 mmol) in THF (0.6 mL) at 0° C. After 5min, 4-hydroxy-2-(2,6-dioxo(3-piperidyl))isoindoline-1,3-dione (550 mg,2.0 mmol) was added to the mixture. The mixture was allowed to warm toroom temperature and kept for 4 h. The solvent was removed in vacuo, andthe residue was purified by column chromatography (Silca Gel) to give anoil. The oil was slurried in methanol (10 mL) for 3 h to give asuspension. The suspension was filtered and washed with methanol (20 mL)to give2-(2,6-dioxo-piperidin-3-yl)-4-(furan-2-ylmethoxy)-isoindole-1,3-dioneas a yellow solid (310 mg, 44% yield): mp, 184-186° C.; ¹H NMR (DMSO-d₆)δ 1.99-2.04 (m, 1H, CHH), 2.42-2.61 (m, 2H, CH₂), 2.80-2.95 (m, 1H,CHH), 5.07 (dd, J=5, 13 Hz, 1H, NCH), 5.35 (s, 2H, CH₂), 6.49 (dd, J=2,3 Hz, 1H, Ar), 6.68 (d, J=8 Hz, 1H, Ar), 7.47 (d, J=7 Hz, 1H, Ar),7.68-7.71 (m, 2H, Ar), 7.83 (t, J=8 Hz, 1H, Ar), 11.10 (s, 1H, NH); ¹³CNMR (DMSO-d₆) δ 21.96, 30.93, 48.77, 62.55, 110.72, 111.38, 115.72,116.64, 120.35, 133.35, 136.89, 143.99, 149.15, 155.12, 165.21, 166.73,169.88, 172.76; Anal Calcd for C₁₈H₁₄N₂O₆+0.1H₂O: C, 60.71; H, 4.02; N,7.87; H₂O, 0.51. Found: C, 60.47; H, 3.97; N, 7.73; H₂O, 0.38.

Example 204-(3-Chloro-benzo[b]thiophen-2-ylmethoxy)-2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dione

Step 1:

To a solution of 3-chloro-benzo[b]thiophene-2-carboxylic acid (3.5 g,16.6 mmol) in THF (40 mL) at 0° C. was added drop-wise 1 M borane in THF(33 mL, 33.2 mmol) via a dropping funnel. The mixture was stirred at r.tovernight. The reaction was quenched with drop-wise addition of water (6mL). Solvent was evaporated in vacuo and the residue was partitionedbetween sat. sodium carbonate and ethyl acetate. The aqueous phase wasextracted with ethyl acetate (100 mL), the combined organic phases waswashed with water (3×100 mL), dried and concentrated to give(3-chloro-benzo[b]thiophen-2-yl)-methanol as a light yellow solid (3.4g, 103% crude yield); ¹H NMR (DMSO-d₆) δ 4.8 (d, J=5.8 Hz, 2H, CH₂OH),5.87 (t, J=5.8 Hz, 1H, CH₂OH), 7.42-8.04 (m, 4H, Ar). The product wasused in the next step without further purification.

Step 2:

To a stirred suspension of 3-hydroxyphthalic acid dimethyl ester (1.0 g,4.8 mmol), (3-chloro-benzo[b]thiophen-2-yl)-methanol (1.9 g, 9.5 mmol),and polymer-supported triphenyl phosphine (3.0 g, 9.5 mmol) in THF (30mL) in an ice-bath was slowly added diisopropyl azodicarboxylate (1.9mL, 9.5 mmol) and stirred at r.t. overnight. The mixture was filteredand the solid was washed with ethyl acetate (10 mL). The filtrate wasevaporated and the residue was purified by flash column chromatography(EtOAc/Hexane) to give3-(3-chloro-benzo[b]thiophen-2-ylmethoxy)-phthalic acid dimethyl ester(2.1 g, 109% crude yield). The product was used in the next step withoutfurther purification.

Step 3:

A solution of 3-(3-chloro-benzo[b]thiophen-2-ylmethoxy)-phthalic aciddimethyl ester (1.9 g, 4.8 mmol) in reagent alcohol (120 mL) and 3 Nsodium hydroxide (60 mL) was refluxed for two hours. The solution wasevaporated and the residue was dissolved in water (100 mL) and washedwith methylene chloride (3×100 mL) then acidified to pH around 4. Theresulting mixture was extracted with ethyl acetate (2×100 mL) and thecombined organic layers was washed with water (2×100 mL), dried andconcentrated to give 3-(3-chloro-benzo[b]thiophen-2-ylmethoxy)-phthalicacid as an off-white solid (1.6 g, 92% yield). The product was used inthe next step without further purification.

Step 4:

A mixture of 3-(3-chloro-benzo[b]thiophen-2-ylmethoxy)-phthalic acid(1.6 g, 4.4 mmol), alpha-amino-glutarimide hydrochloride (0.76 g, 4.6mmol) in pyridine was refluxed overnight. The mixture was evaporated andthe residue was purified by flash column chromatography(methanol/methylene chloride) to give4-(3-chloro-benzo[b]thiophen-2-ylmethoxy)-2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dioneas a white solid (0.76 g, 38% yield); HPLC: Waters Symmetry C₁₈, 5 μm,3.9×150 mm, 1 mL/min, 240 nm, 60/40 CH₃CN/0.1% H₃PO₄, 5.26 min (98.7%);mp, 240-242° C.; ¹H NMR (DMSO-d₆) δ 2.02-2.06 (m, 1H, CHH), 2.54-2.62(m, 2H, CH₂), 2.83-2.95 (m, 1H, CHH), 5.10 (dd, J=6, 12 Hz, 1H, CH),5.74 (s, 2H, CH₂), 7.49-8.10 (m, 7H, Ar), 11.12 (s, 1H, NH); ¹³C NMR(DMSO-d₆) δ21.94, 30.90, 48.79, 63.99, 116.26, 116.98, 118.36, 120.46,121.39, 123.36, 125.57, 126.19, 132.93, 133.34, 135.42, 136.91, 137.04,154.62, 165.05, 166.64, 169.85, 172.73. Anal Calcd ForC₂₂H₁₅N₂O₅SCl+0.1H₂O: C, 58.09; H, 3.32; N, 6.16; S, 7.05; Cl, 7.79.Found: C, 57.77; H, 3.06; N, 6.08; S, 6.87; Cl, 8.05.

Example 212-(2,6-Dioxo-piperidin-3-yl)-4-(4-fluoro-benzo[b]thiophen-2-ylmethoxy)-isoindole-1,3-dione

Step 1:

To a stirred suspension of 3-hydroxyphthalic acid dimethyl ester (1.1 g,5.2 mmol), (4-fluoro-benzo[b]thiophen-2-yl)-methanol (0.96 g, 10.5mmol), and polymer-supported triphenyl phosphine (3.0 g, 10.5 mmol) inTHF (35 mL) in an ice-bath was slowly added diisopropyl azodicarboxylate(2.1 mL, 10.5 mmol) and stirred at r.t. overnight. The mixture wasfiltered and the solid was washed with ethyl acetate (10 mL). Thefiltrate was evaporated and the residue was purified by flash columnchromatography (EtOAc/Hexane) to give3-(4-fluoro-benzo[b]thiophen-2-ylmethoxy)-phthalic acid dimethyl ester(1.5 g, 76% yield). The product was used in the next step withoutfurther purification.

Step 2:

A solution of 3-(4-fluoro-benzo[b]thiophen-2-ylmethoxy)-phthalic aciddimethyl ester (1.5 g, 4.0 mmol) in reagent alcohol (120 mL) and 3 Nsodium hydroxide (60 mL) was refluxed for two hours. The solution wasevaporated and the residue was dissolved in water (100 mL) and washedwith methylene chloride (3×100 mL) then acidified to pH around 4. Theresulting mixture was extracted with ethyl acetate (2×100 mL) and thecombined organic layers was washed with water (2×100 mL), dried andconcentrated to give 3-(4-fluoro-benzo[b]thiophen-2-ylmethoxy)-phthalicacid as an off-white solid (1.2 g, 84% yield). The product was used inthe next step without further purification.

Step 3:

A mixture of 3-(4-fluoro-benzo[b]thiophen-2-ylmethoxy)-phthalic acid(1.2 g, 3.4 mmol), alpha-amino-glutarimide hydrochloride (0.58 g, 3.6mmol) in pyridine was refluxed overnight. The mixture was evaporated andthe residue was purified by flash column chromatography(methanol/methylene chloride) to give2-(2,6-dioxo-piperidin-3-yl)-4-(4-fluoro-benzo[b]thiophen-2-ylmethoxy)-isoindole-1,3-dioneas a white solid (0.66 g, 44% yield); HPLC: Waters Symmetry C₁₈, 5 μm,3.9×150 mm, 1 mL/min, 240 nm, 60/40 CH₃CN/0.1% H₃PO₄, 3.08 min (97.5%);mp, 264-266° C.; ¹H NMR (DMSO-d₆) δ 2.01-2.08 (m, 1H, CHH), 2.54-2.95(m, 2H, CHHCH₂), 5.11 (dd, J=6, 12 Hz, 1H, CH), 5.73 (s, 2H, CH₂),7.18-7.88 (m, 7H, Ar), 11.12 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ 21.98,30.95, 48.82, 65.85, 109.57, 109.81, 116.04, 116.91, 118.02, 119.02,119.07, 120.37, 125.98, 126.08, 127.39, 127.65, 133.38, 137.02, 140.81,141.98, 142.06, 154.81, 155.10, 158.41, 165.17, 166.71, 169.91, 172.78.Anal Calcd For C₂₂H₁₅N₂O₅SF: C, 60.27; H, 3.45; N, 6.39; S, 7.31; F,4.33. Found: C, 60.40; H, 3.26; N, 6.29; S, 7.24; F, 4.32.

Example 224-(5-Chloro-thiophen-2-ylmethoxy)-2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dione

Step 1:

To a stirred suspension of 3-hydroxyphthalic acid dimethyl ester (1.4 g,6.8 mmol) in acetone (70 mL) and potassium carbonate (2.8 g, 20 mmol)was added 2-chloro-5-chloromethyl-thiophene (0.83 mL, 7.1 mmol) andrefluxed for two hours. The solvent was evaporated and the residue waspartitioned between water (100 mL) and ethyl acetate (150 mL) and washedwith water (2×100 mL). The combined organic phases was dried,concentrated and purified by flash column chromatography (EtOAc/Hexane)to give 3-(5-chloro-thiophen-2-ylmethoxy)-phthalic acid dimethyl ester(2.3 g, 100% crude yield). The product was used in the next step withoutfurther purification.

Step 2:

A solution of 3-(5-chloro-thiophen-2-ylmethoxy)-phthalic acid dimethylester (2.3 g, 6.7 mmol) in reagent alcohol (100 mL) and 3 N sodiumhydroxide (60 mL) was refluxed for two hours. The solution wasevaporated and the residue was dissolved in water (100 mL) and washedwith methylene chloride (3×100 mL) then acidified to pH around 4. Theresulting mixture was extracted with ethyl acetate (2×100 mL) and thecombined organic layers was washed with water (2×100 mL), dried andconcentrated to give 3-(5-chloro-thiophen-2-ylmethoxy)-phthalic acid asan off-white solid (1.6 g, 76% yield). The product was used in the nextstep without further purification.

Step 3:

A mixture of 3-(5-chloro-thiophen-2-ylmethoxy)-phthalic acid (1.6 g, 5.1mmol), alpha-amino-glutarimide hydrochloride (0.88 g, 5.4 mmol) inpyridine was refluxed overnight. The mixture was evaporated and theresidue was purified by flash column chromatography (methanol/methylenechloride) to give4-(5-chloro-thiophen-2-ylmethoxy)-2-(2,6-dioxo-piperidin-3-yl)-isoindole-1,3-dioneas a white solid (0.76 g, 36% yield); HPLC: Waters Symmetry C₁₈, 5 μm,3.9×150 mm, 1 mL/min, 240 nm, 60/40 CH₃CN/0.1% H₃PO₄, 2.63 min (99.3%);mp, 217-219° C.; ¹H NMR (DMSO-d₆) δ 2.01-2.07 (m, 1H, CHH), 2.54-2.57(m, 2H, CH₂), 2.62-2.95 (m, 1H, CHH), 5.10 (dd, J=6, 12 Hz, 1H, CH),5.50 (s, 2H, CH₂), 7.07-7.87 (m, 5H, Ar), 11.11 (s, 1H, NH); ¹³C NMR(DMSO-d₆) δ 21.93, 30.90, 48.75, 65.38, 115.94, 116.80, 120.44, 126.55,127.83, 129.09, 133.31, 136.93, 137.54, 154.76, 165.14, 166.68, 169.77,169.86, 172.73. Anal Calcd For C₁₈H₁₃N₂O₅SCl: C, 53.41; H, 3.24; N,6.92; S, 7.92%; Cl, 8.76. Found: C, 53.39; H, 2.95; N, 6.80; S, 7.62%;Cl, 9.01.

Example 232-(2,6-Dioxo-piperidin-3-yl)-4-(1-naphthalen-2-yl-ethoxy)-isoindole-1,3-dione

Step 1:

A mixture of polymer-supported PPh₃ (3.1 g, ˜9.5 mmol) andα-methyl-2-naphthalenemethanol (0.82 g, 4.8 mmol) in 20 mL THF wascooled to 0° C. under N₂. Diisopropylazodicarboxylate (1.9 g, 9.5 mmol)was added dropwise, and subsequently 3-hydroxy-phthalic acid dimethylester (1.0 g, 4.8 mmol) was added as a solid. The mixture stirred for anadditional hour at 0° C. and was then allowed to warm to roomtemperature. After stirring for 16 h, the mixture was filtered. Thefilter was washed with ethyl acetate (20 mL) and the combined filtrateswere evaporated. The residue was chromatographed in hexanes-ethylacetate gradient, eluting 1.2 g of the3-(1-naphthalen-2-yl-ethoxy)-phthalic acid dimethyl ester at 20-30%ethyl acetate, in 66% yield; ¹H NMR (DMSO-d₆) δ 1.70 (d, J=6.5 Hz, 3H),3.88 (s, 3H), 4.03 (s, 3H), 5.49 (q, J=6.5 Hz, 1H), 6.96 (d, J=8.4 Hz,1H), 7.18 (t, J=8.0 Hz, 1H), 7.42-7.53 (m, 4H), 7.76-7.84 (m, 4H).

Step 2:

A mixture of 3-(1-naphthalen-2-yl-ethoxy)-phthalic acid dimethyl ester(0.9 g, 2.5 mmol) and 3N NaOH (50 mL) in ethanol (100 mL) was heated toreflux for 2 h. The mixture was cooled and the solvent was removed undervacuum. The residue was dissolved in water (100 mL) and washed withCH₂Cl₂ (3×100 mL), acidified (HCl), and extracted with ethyl acetate(3×75 mL). The combined organic extracts were washed with water (3×75mL), dried (MgSO₄), and evaporated, providing 0.50 g of3-(1-naphthalen-2-yl-ethoxy)-phthalic acid, in 60% yield; ¹H NMR(DMSO-d₆) δ 1.60 (d, J=6.2 Hz, 3H), 5.79 (q, J=6.2 Hz, 1H), 7.21-7.31(m, 2H), 7.38 (dd, J=7.1 Hz, J=1.3 Hz, 1H), 7.46-7.57 (m, 3H), 7.83-7.93(m, 4H).

Step 3:

A mixture of 3-(1-naphthalen-2-yl-ethoxy)-phthalic acid (0.36 g, 1.0mmol) and rac-α-aminoglutarimide hydrochloride (0.16 g, 1.0 mmol) inpyridine (10 mL) was heated to reflux for 16 h. The mixture was cooledand evaporated under vacuum. The residue was dissolved in ethyl acetate(100 mL) and washed with dilute aqueous HCl (2×100 mL) and water (100mL), and was evaporated. The residue was chromatographed using aCH₂Cl₂-methanol gradient, eluting the product at 95:5 CH₂Cl₂-methanol,0.27 g, in 64% yield; mp 174-176° C.; HPLC, Waters Symmetry C-18,3.9×150 mm, 5 μm, 1 mL/min, 240 nm, 60/40 CH₃CN/0.1% H₃PO₄, 3.69(99.65%); ¹H NMR (DMSO-d₆) δ 1.71 (d, J=6.0 Hz, 3H), 1.99-2.09 (m, 1H),2.51-2.65 (m, 2H), 2.84-2.97 (m, 1H), 5.13 (dd, J=12.5 Hz, J=5.3 Hz,1H), 6.00 (q, J=6.0 Hz, 1H), 7.39 (d, J=7.2 Hz, 1H), 7.44-7.53 (m, 3H),7.62 (d, J=8.5 Hz, 1H), 7.69 (t, J=7.9 Hz, H), 7.87-7.96 (m, 3H), 8.00(s, 1H), 11.15 (s, 1H); ¹³C NMR (DMSO-d₆) δ 22.0, 23.7, 31.0, 48.8,76.5, 115.5, 117.2, 121.5, 123.6, 124.5, 126.2, 126.4, 127.6, 127.8,128.5, 132.5, 132.7, 133.4, 136.7, 139.4, 154.8, 165.3, 166.7, 170.0,172.8; Anal. Calcd for C₅H₂₀N₂O₅: C, 70.08; H, 4.71; N, 6.54. Found: C,69.71; H, 4.51; N, 6.28.

Example 242-(2,6-Dioxo-piperidin-3-yl)-4-(4-methoxy-benzyloxy)-isoindole-1,3-dione

Step 1:

A stirred mixture of 3-hydroxyphthalic anhydride (20.5 g, 125 mmol) inmethanol (100 mL) was heated to reflux for three hours. The solvent wasevaporated in vacuo, and the residue was suspended in sodium bicarbonate(29.4 g, 350 mmol) in DMF (250 mL), followed by addition of iodomethane(19 mL, 300 mmol) and heating at 55° C. for four hours. The mixture wascooled to room temperature, solvent evaporated in vacuo, and the residuewas partitioned between water (200 mL) and ethyl acetate (200 mL). Theorganic layer was washed with water (2×200 mL), dried, concentrated invauco, and then purified by flash column chromatography (Silica Gel,EtOAc/Hexane, 0% gradient to 100% 30 min) to give 3-hydroxyphthalic aciddimethyl ester (20.2 g, 77% yield). The product was used in the nextstep without further purification.

Step 2:

To a stirred suspension of 3-hydroxyphthalic acid dimethyl ester (1.1 g,5.2 mmol) in acetone (45 mL) and potassium carbonate (2.2 g, 15.7 mmol),was added 1-bromomethyl-4-methoxy-benzene (0.79 mL, 5.5 mmol). Themixture was refluxed for six hours. The solvent was evaporated in vacuo,and the residue was partitioned between water (50 mL) and ethyl acetate(80 mL). The organic layer was washed with water (2×50 mL), dried,concentrated in vacuo, and purified by flash column chromatography(Silica Gel, EtOAc/Hexane, 0% gradient to 100% 30 min) to give3-(4-methoxy-benzyloxy)-phthalic acid dimethyl ester (2.0 g, 115% crudeyield). The product was used in the next step without furtherpurification.

Step 3:

A stirred solution of 3-(4-methoxy-benzyloxy)-phthalic acid dimethylester (2.0 g crude, 5.5 mmol) in reagent alcohol (100 mL) and 3 N sodiumhydroxide (35 mL) was refluxed for two hours. The solution wasevaporated in vacuo, and the residue was dissolved in water (80 mL) andwashed with methylene chloride (3×70 mL), then acidified to pH around 4by HCl. The resulting mixture was extracted with ethyl acetate (2×60mL), and the combined organic layers was washed with water (2×70 mL),dried, and concentrated in vacuo to give3-(4-methoxy-benzyloxy)-phthalic acid as an off-white solid (1.6 g, 100%crude yield). The product was used in the next step without furtherpurification.

Step 4:

A stirred mixture of 3-(4-methoxy-benzyloxy)-phthalic acid (1.5 g, 5.2mmol), alpha-amino-glutarimide hydrochloride (0.90 g, 5.4 mmol) inpyridine was refluxed overnight. The mixture was evaporated in vacuo,and the residue was purified by flash column chromatography (Silica Gel,methanol/methylene chloride, 0% gradient to 10% 30 min) to give2-(2,6-dioxo-piperidin-3-yl)-4-(4-methoxy-benzyloxy)-isoindole-1,3-dioneas a white solid (0.83 g, 40% yield); HPLC: Waters Symmetry C₁₈, 5 μm,3.9×150 mm, 1 mL/min, 240 nm, 50/50 CH₃CN/0.1% H₃PO₄, RT=4.17 min(98.6%); mp, 178-180° C.; ¹HNMR (DMSO-d₆) δ1.99-2.06 (m, 1H, CHH),2.51-2.82 (m, 2H, CHHCH₂), 3.76 (s, 3H, CH₃), 5.08 (dd, J=6, 12 Hz, 1H,CH), 5.29 (s, 2H, CH₂), 6.95-7.84 (m, 7H, Ar), 11.11 (s, 1H, NH); ¹³CNMR (DMSO-d₆) δ 21.96, 30.91, 48.72, 55.08, 69.92, 113.88, 115.41,116.55, 120.29, 127.93, 129.21, 133.25, 136.92, 155.57, 159.10, 165.28,166.77, 169.90, 172.75. Anal Calcd For C₂₁H₁₈N₂O₆: C, 63.96; H, 4.60; N,7.10. Found: C, 63.86; H, 4.30; N, 6.92.

5.1 Assays

5.1.1 TNFα Inhibition Assay in PMBC

Peripheral blood mononuclear cells (PBMC) from normal donors areobtained by Ficoll Hypaque (Pharmacia, Piscataway, N.J., USA) densitycentrifugation. Cells are cultured in RPMI 1640 (Life Technologies,Grand Island, N.Y., USA) supplemented with 10% AB+human serum (GeminiBio-products, Woodland, Calif., USA), 2 mM L-glutamine, 100 U/mlpenicillin, and 100 μg/ml streptomycin (Life Technologies).

PBMC (2×10⁵ cells) are plated in 96-well flat-bottom Costar tissueculture plates (Corning, N.Y., USA) in triplicate. Cells are stimulatedwith LPS (from Salmonella abortus equi, Sigma cat. no. L-1887, St.Louis, Mo., USA) at 1 ng/ml final in the absence or presence ofcompounds. Compounds provided herein are dissolved in DMSO (Sigma) andfurther dilutions are done in culture medium immediately before use. Thefinal DMSO concentration in all assays can be about 0.25%. Compounds areadded to cells 1 hour before LPS stimulation. Cells are then incubatedfor 18-20 hours at 37° C. in 5% CO₂, and supernatants are thencollected, diluted with culture medium and assayed for TNFαc levels byELISA (Endogen, Boston, Mass., USA). IC₅₀s are calculated usingnon-linear regression, sigmoidal dose-response, constraining the top to100% and bottom to 0%, allowing variable slope (GraphPad Prism v3.02).

5.1.2 IL-2 and MIP-3α Production by T Cells

PBMC are depleted of adherent monocytes by placing 1×10⁸ PBMC in 10 mlcomplete medium (RPMI 1640 supplemented with 10% heat-inactivated fetalbovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/mlstreptomycin) per 10 cm tissue culture dish, in 37° C., 5% CO₂ incubatorfor 30-60 minutes. The dish is rinsed with medium to remove allnon-adherent PBMC. T cells are purified by negative selection using thefollowing antibody (Pharmingen) and Dynabead (Dynal) mixture for every1×10⁸ non-adherent PBMC: 0.3 ml Sheep anti-mouse IgG beads, 15 μlanti-CD16, 15 μl anti-CD33, 15 μl anti-CD56, 0.23 ml anti-CD19 beads,0.23 ml anti-HLA class II beads, and 56 μl anti-CD14 beads. The cellsand bead/antibody mixture is rotated end-over-end for 30-60 minutes at4° C. Purified T cells are removed from beads using a Dynal magnet.Typical yield is about 50% T cells, 87-95% CD3⁺ by flow cytometry.

Tissue culture 96-well flat-bottom plates are coated with anti-CD3antibody OKT3 at 5 μg/ml in PBS, 100 μl per well, incubated at 37° C.for 3-6 hours, then washed four times with complete medium 100 μl/welljust before T cells are added. Compounds are diluted to 20 times offinal in a round bottom tissue culture 96-well plate. Finalconcentrations are about 10 μM to about 0.00064 μM. A 10 mM stock ofcompounds provided herein is diluted 1:50 in complete for the first 20×dilution of 200 μM in 2% DMSO and serially diluted 1:5 into 2% DMSO.Compound is added at 10 μl per 200 μl culture, to give a final DMSOconcentration of 0.1%. Cultures are incubated at 37° C., 5% CO₂ for 2-3days, and supernatants analyzed for IL-2 and MIP-3α by ELISA (R&DSystems). IL-2 and MIP-3α levels are normalized to the amount producedin the presence of an amount of a compound provided herein, and EC₅₀scalculated using non-linear regression, sigmoidal dose-response,constraining the top to 100% and bottom to 0%, allowing variable slope(GraphPad Prism v3.02).

5.1.3 Cell Proliferation Assay

Cell lines Namalwa, MUTZ-5, and UT-7 are obtained from the DeutscheSammlung von Mikroorganismen and Zellkulturen GmbH (Braunschweig,Germany). The cell line KG-1 is obtained from the American Type CultureCollection (Manassas, Va., USA). Cell proliferation as indicated by³H-thymidine incorporation is measured in all cell lines as follows.

Cells are plated in 96-well plates at 6000 cells per well in media. Thecells are pre-treated with compounds at about 100, 10, 1, 0.1, 0.01,0.001, 0.0001 and 0 μM in a final concentration of about 0.25% DMSO intriplicate at 37° C. in a humidified incubator at 5% CO₂ for 72 hours.One microcurie of ³H-thymidine (Amersham) is then added to each well,and cells are incubated again at 37° C. in a humidified incubator at 5%CO₂ for 6 hours. The cells are harvested onto UniFilter GF/C filterplates (Perkin Elmer) using a cell harvester (Tomtec), and the platesare allowed to dry overnight. Microscint 20 (Packard) (25 μl/well) isadded, and plates are analyzed in TopCount NXT (Packard). Each well iscounted for one minute. Percent inhibition of cell proliferation iscalculated by averaging all triplicates and normalizing to the DMSOcontrol (0% inhibition). Each compound is tested in each cell line inthree separate experiments. Final IC₅₀s are calculated using non-linearregression, sigmoidal dose-response, constraining the top to 100% andbottom to 0%, allowing variable slope. (GraphPad Prism v3.02).

5.1.4 Immunoprecipitation and Immunoblot

Namalwa cells are treated with DMSO or an amount of a compound providedherein for 1 hour, then stimulated with 10 U/ml of Epo (R&D Systems) for30 minutes. Cell lysates are prepared and either immunoprecipitated withEpo receptor Ab or separated immediately by SDS-PAGE. Immunoblots areprobed with Akt, phospho-Akt (Ser473 or Thr308), phospho-Gab1 (Y627),Gab1, IRS2, actin and IRF-1 Abs and analyzed on a Storm 860 Imager usingImageQuant software (Molecular Dynamics).

5.1.5 Cell Cycle Analysis

Cells are treated with DMSO or an amount of a compound provided hereinovernight. Propidium iodide staining for cell cycle is performed usingCycleTEST PLUS (Becton Dickinson) according to manufacturer's protocol.Following staining, cells are analyzed by a FACSCalibur flow cytometerusing ModFit LT software (Becton Dickinson).

5.1.6 Apoptosis Analysis

Cells are treated with DMSO or an amount of a compound provided hereinat various time points, then washed with annexin-V wash buffer (BDBiosciences). Cells are incubated with annexin-V binding protein andpropidium iodide (BD Biosciences) for 10 minutes. Samples are analyzedusing flow cytometry.

5.1.7 Luciferase Assay

Namalwa cells are transfected with 4 μg of AP1-luciferase (Stratagene)per 1×10⁶ cells and 3 μl Lipofectamine 2000 (Invitrogen) reagentaccording to manufacturer's instructions. Six hours post-transfection,cells are treated with DMSO or an amount of a compound provided herein.Luciferase activity is assayed using luciferase lysis buffer andsubstrate (Promega) and measured using a luminometer (Turner Designs).

5.1.8 TNFα Inhibition and IL-2 Production

Using procedures substantially similar to those provided in Section5.1.1 above, IC₅₀ values for certain of the compounds provided hereinfor TNFα inhibition were determined. The determined IC₅₀ values rangedfrom less than 0.2 nM to about 10-100 μM. These results show thatcompounds provided herein are useful as inhibitors of TNFα.

Using procedures substantially similar to those described in Section5.1.2. above, EC₅₀ values of certain compounds provided herein for theproduction of IL-2 were also determined. The determined EC₅₀ valuesranged from greater than 1 nM to less than 1 μM. These results show thatcompounds provided herein are useful as stimulators of IL-2 production.

The embodiments described above are intended to be merely exemplary, andthose skilled in the art will recognize, or will be able to ascertainusing no more than routine experimentation, numerous equivalents ofspecific compounds, materials, and procedures. All such equivalents areconsidered to be within the scope of the claimed subject matter and areencompassed by the appended claims.

All of the patents, patent applications and publications referred toherein are incorporated herein in their entireties. Citation oridentification of any reference in this application is not an admissionthat such reference is available as prior art to the claimed subjectmatter. The full scope of the invention is better understood withreference to the appended claims.

What is claimed is:
 1. A compound of formula:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:Y is C═O or CH₂; and R¹ is alkyl substituted with one or more groupsselected from alkoxy, halo, carboxy, alkylaminocarbonyl, alkoxycarbonyl,nitro, nitrile, haloalkyl, hydroxy, and alkylsulfonyl.
 2. The compoundof claim 1, wherein Y is C═O.
 3. The compound of claim 1, wherein Y isCH₂.
 4. The compound of claim 1, wherein the compound is:


5. A pharmaceutical composition comprising a compound of claim 1, or apharmaceutically acceptable salt, or stereoisomer thereof.